S1

S1
Basic Biochemical Roots
Dietrich H. Nies

S1.1 Chemistry and Physics of Life S2 S1.2.10 The F1 F0 -ATPase S21

S1.1.1 Thermodynamics of Life S2 S1.2.11 Energy Pools in the Cell S21
S1.1.2 The Three Levels of Energy Transformation in S1.2.12 Life Styles S22
Living Cells S3
S1.1.3 Macromolecules S4 S1.3 Basic Biochemistry S26
S1.1.4 Necessity of a Semipermeable Membrane S4 S1.3.1 Organization of the Overall Metabolism S26
S1.1.5 Chemical Elements Available for Life S5 S1.3.2 Enzymes and Coenzymes S27
S1.1.6 Solvents of Life S6 S1.3.3 The Backbone: Fructose-1,6-bP Pathway S29
S1.3.4 Cycles and Shunts Attached to the
S1.2 Energy and Transport S8 F1,6bP-Pathway S33
S1.2.1 Energy, Chemical Elements, and S1.3.5 Fates of Pyruvate S38
Macromolecules S8 S1.3.6 Fates of Acetyl-S-CoA S40
S1.2.2 Atoms and Orbitals S9 S1.3.7 Putting It Together: Anaerobic
S1.2.3 Redox Energy and Electronegativity S11 Ecosystems S44
S1.2.4 Atoms in Molecules S12 S1.3.8 Assimilation of the 10 Macrobioelements S46
S1.2.5 Functional Groups and Energy-Rich S1.3.9 Building Blocks S48
Bonds S13 S1.3.10 Macromolecules in Bacteria S52
S1.2.6 Energy Sources for Life: Light Energy S15 S1.3.11 DNA-Replication and Cell Division in
S1.2.7 Hierarchy of Transport Processes S16 Prokaryots S55
S1.2.8 Ion Motive Forces S18 S1.3.12 Genomes and Evolution S56
S1.2.9 How to Built up a Proton Motive Force by References S60
Redox Energy S19 Further Reading S60

Ecological Biochemistry: Environmental and Interspecies Interactions, First Edition. Edited by Gerd-Joachim Krauss and Dietrich H. Nies.
2015 Wiley-VCH Verlag GmbH & Co. KGaA. Published 2015 by Wiley-VCH Verlag GmbH & Co. KGaA.
Companion Website: http://www.wiley.com/go/Krauss/Nies/EcologicalBiochemistry
S2 S1 Basic Biochemical Roots
S1.1
Chemistry and Physics of Life
Overview
How does life function? This chapter starts explaining
the thermodynamics of life, shows that living entities are
no exceptions from the important laws of thermodynam-
ics, and derives the physical and chemical constraints of
the life process from here. Moreover, the basics of energy
S1.1.1
Thermodynamics of Life
Every year in spring the wonder happens. When the tem-
perature turns milder, owers explode from seeds or bulbs
in the ground, and leafs appear on bushes and trees. On rst
glance, order seems to be spontaneous. However, this spon-
taneous appearance of order is against the laws of thermo-
dynamics. Therefore, people thought in previous times that
this appearance of order in living organisms was allowed by
a special life force.
The word thermodynamics comes from Greek ther-
ms = warm and dynamis = power. The principles of
thermodynamics were formulated in the eighteenth cen-
tury to improve the eciency of steam engines, which were
in fact already known as aeolipiles by the Greek engineer
and mathematician Hero of Alexandria (about 1070 AD).
James Watt (17361819) pioneered a major improvement
of the steam engine in the eighteenth century but many
other scientists, such as Otto von Guericke, Robert Boyle,
Robert Hooke, Joseph Black, Sadi Carnot, and William
Rankine, also performed experimental work that led to
the development of the principles of thermodynamics.
transduction, the four most important classes of cellular
macromolecules, the necessity of a semipermeable mem-
brane, the chemical components and solvents of life are
introduced.
This paved way for the industrial revolution in Europe and
elsewhere, with all its social and political consequences.
The rst law of thermodynamics states that energy cannot
be created or destroyed. It is just that one form of energy
can be transformed into another form of energy. This may
be an unwelcome fact in times of high prizes for fuel; how-
ever, a spontaneous loss of energy would allow disappear-
ance of matter such as this book or even its reader. Taking
this aspect into account, the rst law of thermodynamics
makes our universe a much safer place.
The two other laws have to do with entropy, which
means disorder. The second law states that a process
occurs spontaneously only if the entropy of the respective
system increases. A writing desk in an oce is a good
illustration of this principle, childrens rooms are even
better ones. The third law indicates that a system cannot
be transferred into a state that does not contain any energy
because it would have no entropy under these conditions.
As a consequence, a temperature of 0 K cannot be reached.
So, how do plants that sprout in spring especially cope
with the second law of thermodynamics? A spontaneous
appearance of order should not be allowed.
This contradiction was explained by Erwin Schrdinger
(18871961) in his famous lecture What is life? delivered

at Trinity College, Dublin, in February 1943 and published
in 1944 for the rst time. This lecture connects biology
rmly to chemistry and physics by showing that life also
obeys thermodynamics, and it was one of the theoretical
foundations of modern molecular biology.
Life is a chemical process that is essentially connected
to compartments separated from their environment, cells.
These cells have to take up energy continuously to keep their
state of order within, named negentropy by Schrdinger.
To cope with the second law of thermodynamics, cells
compensate the increase of order inside by a stronger
decrease of order outside the cell. So, the decrease of the
entropy inside is overcompensated by the increase of the
entropy outside. Dened as one system, cells and their
environment continuously enhance the entropy, following
the second law of thermodynamics but allowing an increase
of order inside the cell at the same time. This process can be
driven by external energy or energy forms previously stored
(Figure S1.1).
Very important, these compartments, the cells, are
needed to make a dierence between the state of order
inside and outside. The exact phrasing of this connection
by Schrdinger was Thus a living organism continually
increases its entropy or, as you may say, produces positive
entropy and thus tends to approach the dangerous state
of maximum entropy, which is of death. It can only keep
aloof from it, i.e. alive, by continually drawing from its
environment negative entropy which is something very
positive as we shall immediately see. What an organism
feeds upon is negative entropy. Thus, during the chemical
life process, energy is turned into negentropy and all cells
are continuously extracting order from their environments
(Figure S1.1).
S1.1.2
The Three Levels of Energy Transformation in Living Cells
The transformation of energy into negentropy by living cells
happens on three dierent time levels. First and as quoted
above, continuous transformation of energy from the out-
side or previously stored energy is essential to keep a cell
Box S1.1: The growth rate of a cell depends on its radius
The energy needed for growth Eg depends on the mass
m of the cell, which is connected to its volume V by the
density of living matter, = m/V . For a sphere-like cell,
its volume calculates from its radius V = 4/3 r3 and,
therefore, is Eg proportional to 4/3r3 . The ow of r2 )/(c4/3r3 )(aPm )/(c4/3r3 ) = (ab3)/(cr)
energy is a power (Energy/time) in terms of physics and
depends on the surface of a cell (A = 4r2 ). Some of the
energy that is being transformed per time unit has to be
continuously used for the maintenance energy, so some
maintenance power Pm is always needed. The power
available for growth is the power taken up Pup minus
S1.1 Chemistry and Physics of Life S3
Energy
Cell
Entropy decreases
Entropy increases
Entropy increases strongly
(release of waste products, heat)
Figure S1.1 What is life? To build order inside an organism, cells
continuously use energy to decrease the intracellular entropy (or
increase the intracellular negentropy = order) and overcompensate this
by increasing the entropy in the environment by the release of waste
products and heat. Thus, in the total system composed of a cell and its
environment, the entropy increases steadily during the chemical reac-
tions in a cell, and the second law of thermodynamics is kept. Energy
can be light energy or chemical energy (see Section S1.2). Intracel-
lular order means macromolecules. (Earth photo: Courtesy of NASA.)
(Graphics: D. Dobritzsch.)
from dying. The amount of energy needed to be kept alive
is the maintenance energy, maintenance energy divided by
time the maintenance power. The latter is the minimum
energy ow needed by a cell to survive. Some organisms,
such as autochthonous microorganism, are specialized to
survive at a very low maintenance power.
Second, if more energy can be taken up from the envi-
ronment than needed for maintenance, cells are allowed to
grow and divide when they too are big. The growth rate of
a cell depends on a constant divided by the radius (half of
the diameter) of a cell (Box S1.1). Thus, a cell with a diam-
eter of 1 m can divide 10 times more rapidly than a cell
with a diameter of 10 m (1 m = 1/1000 mm). This is true
if no substantial amount of the imported energy is needed
for maintenance. In contrast to autochthonous microorgan-
isms, allochthonous microorganisms are adapted to higher
maintenance power Pm . Therefore, the growth rate of
a living cell is directly proportional to (Pup Pm )/Eg :
= a(Pup Pm )/Eg = a(bAPm )/(cm) = a(b4r2 Pm )/
(c4/3r3 ) = (ab4r2 aPm )/(c4/3r3 ) = (ab4
(aPm )/(c4/3r3 ) = (ab3)/(cr)(aPm )/(c4/3r3 )
= 3ab/(c) r1 3a/(4c). Pm r3 = k 1 r1 k 2 Pm r3
with k 1 = 3ab/(c), k 2 = 3a/(4c), and unknown con-
stants a,b,c. Because under most circumstances k 1 r1
k 2 Pm r3 the result is = k 1 r1 .
S4 S1 Basic Biochemical Roots
energy inux and rapid growth rates, living thus on the sec-
ond level of energy transformation.
The third level of energy transformation starts when a
cell has divided. The two daughter cells start to compete
with each other for the energy in the environment. If they
are not exactly identical because a mutation has happened
on one of the two cells, one cell performs better than the
other one and is able to divide faster than its sister. In other
words, after the rst division of a cell on Earth, evolution
started as the result of spontaneous mutation, which is
also the direct consequence of the second law of thermo-
dynamics, plus selection as proposed by Charles Darwin
(18091882). Thus, the third level of energy transformation
is evolution.
S1.1.3
Macromolecules
A system is in the maximum state of entropy if it contains
as many particles as possible and if these particles are free to
migrate and rotate in all three dimensions. More precisely,
entropy is proportional to the logarithm of all microstates
of a system. Consequently, negentropy or order means
fewer particles with partly inhibited migration or rotation.
This is nothing else but a description of macromolecules,
which are huge molecules composed of modules or building
blocks, which again comprise many atoms. So, life means
that living cells take up energy from the environment,
transform some of the energy into negentropy inside
the cell by the synthesis of macromolecules, and over-
compensate this process by an increase of the entropy
outside the cell through the release of small waste products
and/or heat.
A special kind of negentropy that can be transmitted,
translated, stored, copied, and/or multiplied is informa-
tion. Some of the macromolecules of a living cell are,
therefore, also involved in storage or procession of infor-
mation, while others are redundant macromolecules. The
second law of thermodynamics also states that there is
danger of gradual loss for any kind of information, and that
energy has to be used continuously to maintain or repair
stored information. This denes the spontaneous change of
the stored information of a cell, the mutation, as a natural
consequence of the second law of thermodynamics, which
Table S1.1 Macromolecules.
Name %dry mass Building blocks
Proteins 50 Amino acids
Nucleic acids 20 Nucleotides or desoxynucleotides
Carbohydrates 20 Monosaccharides
Lipids 10 Glycerol(-phosphate), Fatty Acids
leads to the need of information repair processes in all
kinds of cell, and sets the stage for evolution as outlined
above.
There are four main groups of macromolecules in a cell
(Table S1.1). About half of the dry mass of a cell is com-
posed of proteins, which catalyze enzymatic reaction, have
structural, transport, or regulatory functions. Nucleic acids
(RNA, DNA) store and maintain information, and use this
information to govern biosynthesis of proteins. Proteins are
needed for the synthesis of carbohydrates, which could be
constituents of cell walls or storage compounds, or of lipids,
which form biological membranes or are also storage com-
pounds.
S1.1.4
Necessity of a Semipermeable Membrane
Energy transformation and synthesis of macromolecules by
living cells can occur eciently only if cells in general are
in the liquid phase of matter. Life in the gas form should not
allow stable cells and macromolecules. Life in the solid form,
on the other hand, would mean a very slow diusion rate
of the building blocks needed for macromolecule synthesis.
So, solid life would have an extremely slow growth rate and
can be ignored.
Nevertheless, a cell with a liquid interior needs a clearly
dened border to the outside to discriminate increase of
negentropy inside from increase of entropy outside. This
border has to stabilize the cell and prevent its disintegra-
tion, but even more important, the border has to allow
import of energy and export of waste while keeping the
important cellular molecules inside. Therefore, the border
has to contain a semipermeable membrane, which fullls
these requirements. Biological semipermeable membranes
are composed of lipids, which are glycerol-phosphate
units with attached fatty acids or steroid compounds
(Table S1.1).
Control over the kind of particles that are allowed to cross
the semipermeable membrane is mediated by transport
processes (see Section S1.2). This means that transport
processes across the semipermeable membrane are an
important part of the chemical life process. Transport of
most molecules across biological membranes is mediated
by embedded transport proteins.
Function
Transport, regulation, enzymes, structure
Information storage, transcription, translation, regulation
Cell walls and storage
Membranes and storage
 S1.1 Chemistry and Physics of Life S5

S1.1.5 is available, followed by carbon, nitrogen, oxygen, and

Chemical Elements Available for Life
From the point of physics, life process is (i) connected to
cells as reaction compartments that are separated from
their environment by (ii) a semipermeable membrane
allowing (iii) regulated transport processes, which (iv) take
up energy from the environment, use this energy (v) to
increase the negentropy in the liquid inside by (vi) the
synthesis of macromolecules that (vii) may be involved in
information-processing, and which (viii) overcompensate
this by an increase of the entropy in the environment.
Which chemical elements are available in our universe to
mediate such a process?
When the universe came into being at the big bang
13.75 0.11 billion years ago, there was only hydrogen and
some helium. The matter condensed into galaxies compris-
ing stars that produced heat and light by nuclear fusion,
rst of hydrogen to helium, then to carbon, and further
on to iron. The higher the mass of a star is, the brighter it
burns and the shorter it lives. These big ancient stars thus
reached the end of their life rapidly after a billion years or
so, and exploded in spectacular supernova events. That
way, the elements generated in the star were released again
into the environment of the star, and elements of higher
atomic weight than iron were also created. So, with the
exception of the primordial hydrogen and some helium, all
other elements represent the ashes of a long-dead ancient
star.
This mode of element generation also determines the
relative amounts of the chemical elements in the universe.
In general, the higher the atomic weight of an element the
smaller is its frequency of occurrence in a hyperlogarithmi-
cal function. Thus, a huge amount of hydrogen and helium
some alkali and earth alkali metals. Lithium, beryllium, and
boron, however, are present in exceptionally low quantities
because most of them were burned up again during the
element formation process in the ancient stars. On the other
hand, as nuclear fusion is an exergonic (heat-producing)
reaction up to iron, there is also an exceptionally high
content of iron and its neighbors with respect to the high
atomic masses of these transition metals.
So, possible main chemical constituents of a living cell
are the elements H, He, C, N, O plus some metals. With
the exception of chemically inert He, all four elements
are the primary components of the dry mass of living
cells on Earth (Table S1.2), which can be described by
the formula C4.17 H8 O1.25 N1 or shorter C4 H8 (O + N)2.25 or
even shorter <CH2 O> with oxygen standing for itself and
the chemically similar nitrogen. This formula can also be
generalized for any form of life in the universe: to synthesize
a macromolecule in a liquid phase of matter, covalent bonds
between nonmetals are needed (see S1.2 and below).
Not counting T c and Pm , the majority of 68 of the 90
naturally occurring elements are metals. Eight semimetals
show features of a metal and a nonmetal. Sorted according
to their period of the periodic system of the elements, these
are Al/Si, Ge/As, Sb/Te, Po/At with Al, As, Sb, Po being
more metal than nonmetal and Si, Te, Ge the opposite.
Noble gases such as He, Ne, and Ar are chemically inert
and unable to form even small molecules except under
drastic chemical conditions. The halogens F, Cl, Br, I, and
At are able to form one covalent bond only per atom.
This leaves only B, C, N, O of the second period, P and S
of the third, and Se of period four left as real nonmetals
available for formation of macromolecules. These elements
serve as the major bioelements (Table S1.2) or minor

Table S1.2 Major bioelements.

Element %dma) %mdmb) Function

C, carbon 50 4.17 Polymerization core of macromolecules and building blocks

H, hydrogen 8 8 Hydrophobicity of compounds, redox reactions
O, oxygen 20 1.25 Carboxylic, carbonylic and alcoholic groups, sugars
N, nitrogen 14 1.0 Amino groups and heteroaromatic rings
P, phosphor 3.0 0.10 Phosphate residues in nucleic acids, phospholipids and a few proteins
S, sulfur 0.6 0.02 Thiol residues in amino acids and coenzymes
K, potassium 0.6 0.02 Countercation for the negative charges, regulation of osmotic pressure
Mg, magnesium 0.3 0.01 Bridging cation in the cytoplasm, for example, for ATP
Ca, calcium 0.02 0.0005 Bridging cation in membranes
Na, sodium 2.0 0.09 Gradients across the cytoplasmic membrane, pH regulation

a) Percent dry mass.

b) Percent dry mass divided by the atomic mass.
S6 S1 Basic Biochemical Roots

bioelements (Table S1.3), which are also called trace for the formation of a primitive semipermeable membrane.
elements. C-based macromolecules may carry electrically charged
Among the nonmetals, only carbon is able to form stable, side-groups such as amino, carboxylic, or phosphate
multiple CC bonds to polymerize, and to bind also to groups, which allow electrostatic interactions between
H, N, O, S, and Se. Carbon is the ideal element as the macromolecules or parts of one macromolecule. All the
basic constituent of life for a variety of reasons. Its most features cannot be contributed by the neighbors of carbon
oxidized form, carbon dioxide, and its most reduced form, in the periodic system of the elements, B, N, or Si. As
methane, are gases at a broad temperature range and can carbon is also a very important element, most of the life
thus be exchanged between cells across wide distances. forms in our universe should be composed of carbon-based
The oxidation steps in-between both compounds, and macromolecules.
formic acid, formaldehyde, and methanol are also stable
and can be used as building blocks for macromolecules. S1.1.6
Carbon dioxide is soluble in water as carbonate or hydrogen Solvents of Life
carbonate. Carbon-based macromolecules that carry CO
or CN groups are soluble in water while those containing As stated above, life should be possible only in the liquid
exclusively CH groups are not: these compounds are ideal phase of matter. Therefore, a solvent is needed as the
main component of the living cell. Candidates for these
solvents are the hydrogenated forms of the most frequent
Table S1.3 Minor bioelements or trace elements. nonmetals C, N, O: methane CH4 , ammoniac NH3 , and
water H2 O.
Element Atoms/E. colia) Function These rather frequently occurring substances dier in
their polarity, their melting, and boiling points (Table S1.4).
B n.d.b) Quorum sensing, plant cell wall
A high polarity, however, is essential to maintain a
hydrophobic membrane surrounding the interior of the
Cl n.d. Gradients, oxygen-evolving complex of
cell. Therefore, water is by far a better solvent for life than
photosystem II
ammoniac, and methane is not suitable at all.
V n.d. Unorthodox nitrogenases As the rate of a chemical reaction depends on the tem-
Cr n.d. Activation of insulin receptor in perature, the temperature at which these three solvents
mammals remain a liquid has also to be taken into account. The
Mn 12 000 Oxygen-evolving complex of photosystem
Q10 rule states that a reaction rate increases three- to
II, superoxide dismutases, formation of fourfold when the temperature increases by 10 K. As the
bacterial endospores boiling point of ammoniac is 33 C, ammoniac-based
life would be at least 34 = 81 times slower than water-
Fe 290 000 Heme, ironsulfur centers; transfer of
single electrons based life and methane-based life about 50 million-fold.
So, a putative ammoniac-based life that originated only
Co 4500 CC and CH bond rearrangement in
a billion years after the big bang, after the rst ancient
cofactor B12
stars had died, would have evolved into something similar
Ni 9700 Splitting/formation of covalent bonds in to life on Earth after its rst 100 million years: barely
small molecules such as urea, molecular
free-living cells. Thus, ammoniac-based life would be too
hydrogen, methane
slow to make an impact on a planets geochemistry even
Cu 170 000 Reactions with molecular oxygen after several billion years. Moreover, because of the low
Zn 114 000 Non redox-active transition metal polarity of the solvent, ammoniac-based biochemistry
would be much less precise compared to water-based
Se 13 Instead of sulfur in Se-cysteine
biochemistry.
Mo 3450 Orthodox nitrogenases, molybdenum
cofactor-dependent enzymes such as Table S1.4 Possible solvents of life.
nitrate reductase and formiate
dehydrogenases
Compound Polaritya) (%) Melting point ( C) Boiling point ( C)
Cd 192 One example only, carbonic anhydrase
W n.d. Instead of Mo in tungsten
Methane CH4 4 182.5 161.6
cofactor-dependent enzymes such as
formiate dehydrogenases Ammoniac NH3 19 77.7 33
Br, I n.d. Halogenated compounds Water H2 O 39 0 100

a) Numbers per cell (Kirsten et al, 2011). a) Ionic character of the bond to hydrogen as calculated from the
b) Not determined. electronegativities.

So, from the chemical point of view, life is probably
water-based with carbon-based biochemistry. All kinds
of living cells on Earth contain water, usually about
7580%. The mass of living matter containing all its
water is dened as wet mass or wet weight while dry
S1.1 Chemistry and Physics of Life S7
mass or dry weight means the mass after the removal of
the loosely bound water. It should be noted that SI unit
Kg or g refers to a mass and not a weight; however,
this fact is usually ignored in scientic publications and
textbooks.
S8 S1 Basic Biochemical Roots
S1.2
Energy and Transport
Overview
The rst section in this chapter demonstrated how a liv-
ing cell functions in general. In the liquid phase of a sol-
vent, most likely water, a cell represents a separated system
that continuously uses energy from the outside to increase
its negentropy inside by the synthesis of carbon-based
macromolecules, thereby overcompensating the decrease
in entropy inside by a higher increase in entropy outside.
This section shows how this can be accomplished, which
forms of energy a living cell is able to use, and how this
S1.2.1
Energy, Chemical Elements, and Macromolecules
Of the four fundamental interactions in nature (strong
and weak nuclear interaction, electromagnetism, and
gravitation), electromagnetic interaction can be most
readily used by organisms to conserve energy. Gravitation
can be sensed, for instance, by plants but its force is too
small in the m dimension of a living cell to be useful for
the conservation of energy. On the other hand, nuclear
interactions happen on a much smaller scale than that of a
micrometre and are also not useful for life. In an exceptional
situation, however, radioactive decay, a product of nuclear
interactions, produces molecular hydrogen that can be
used by microorganisms in certain environments but this
energy is conserved by the synthesis of macromolecules.
It takes the reader from the fundamental interactions in
nature via atomic orbitals to redox reactions, formation of
molecules from atoms, and to functional groups in macro-
molecules. The basic modes of energy transformation in
living beings is categorized and outlined. Also, transport
processes across biological membranes are explained in a
thorough hierarchical scheme.
is the only and indirect mode of using nuclear forces by a
living cell.
Electromagnetic interactions occur between particles
that contain an electric charge, electrons with a negative
charge and protons with a positive charge. Together with
the electrically neutral neutrons, protons and electrons
form the atoms as the basic constituents of matter. Protons
and neutrons reside in the positively charged atomic
nucleus that has a diameter of about 1 fm (1015 m) and
electrons are located in the negatively charged atomic shell
around the nucleus with a diameter of about 100 pm or
1 (1010 m). In a neutral atom, the number of protons
and electrons is identical and determines the atomic
number of the atom. The number of neutrons is usually
similar or slightly larger to that of the protons. Isotopes

are atoms with the same atomic number but a dierent
number of neutrons. The various isotopes of a chemical
element can be stable or decay radioactively. Protons in
the same atomic nucleus should be repelled from each
other because they have the same electric charge; however,
the strong nuclear interaction, which needs the presence
of neutrons, is stronger at the frequency modulation
scale than the electric interaction and overcomes this
eect.
If an electron moves closer to a positive charge, the
resulting energy is released by a light quantum, a photon.
Photons are thus the exchange particles of the electromag-
netic interaction. If a photon interacts with an electron, the
electron can be moved to a higher state of energy. This is
a useful process for a living cell to conserve energy. Cells
that consume light energy are called phototrophs. On the
other hand, chemotrophs conserve the energy resulting
from a chemical reaction. In chemical reactions, electrons
are transferred from one atom to another (redox energy).
Alternatively, an atom or group of atoms is moved from one
molecule to another and the resulting energy can also be
conserved.
These processes are not as dierent as they appear on
the rst glance. In the light reaction of photosynthesis
used by phototrophs to harvest energy, absorption of
photons is mostly used to increase the redox energy of
cellular compounds. Subsequently, dierences in redox
energy are exploited to form concentration gradients across
a biological membrane. This form of energy is further
transformed into energy-rich chemical bonds and these are
nally needed for the synthesis of macromolecules. So, all
kinds of energy transformation in all the dierent kinds
of organisms on this planet are all on the same roadmap
(Figure S1.2); however, the metabolism of two individual
cells may cover dierent parts of this map.
S1.2.2
Atoms and Orbitals
Atoms are composed of a negatively charged atomic shell
that contains one or more electrons, and about 100 000-fold
smaller positively charged atomic nucleus with protons
and neutrons. The electromagnetic interaction between
electrons and protons keep the electrons in the atom. The
closer they are to the nucleus, the lower is their energy. So,
why do the electrons not move into the atomic nucleus at
all and are done with it? The answer is in Box S2.1.
Electrons are leptons and thus forbidden to have the same
state in the same system, meaning that all the electrons of
an atom must be dierent. Two electrons can dier in their
spin, which describes the direction of rotation of an elec-
tron. The electron rotates either forward (+1/2) or backward
(1/2), but these two possibilities are not sucient to pro-
vide dierent states to all the electrons of an atom. Two elec-
trons that dier only in their spin states but reside in the
same state nevertheless are called an electron pair. These can
S1.2 Energy and Transport S9
Sun
light
h
Photon Phototrophs
(use of light)
Bacteria (photolithoautotrophs,
photoheterotrophs, photomixotrophs)
Halobacterium Plants
Exciton
Chemolithoautotrophs
(use of inorganic compounds)
Redox potential
Chemoorganoheterotrophs
(respiring)
Ion motive force
Energy-rich bond Chemoorganoheterotrophs
(fermenting)
Figure S1.2 The universal roadmap of energy conservation. Pho-
totrophs use light energy (photons) to create an exciton that is sub-
sequently used to change the redox potential of a redox carrier to a
lower potential (= higher energy). Electron transport from the result-
ing low redox potential to a more positive one drives ion transport to
form an ion motive force, mostly in the form of a pmf. Finally, the ion
motive force is used to generate a compound containing an energy-
rich bond such as ATP. To be brief, some archaea such as Halobac-
terium use a protein named bacteriorhodopsin to create an ion motive
force directly from an exciton. Chemolithoautotrophic (cla) bacteria use
the dierence in redox potential of inorganic compounds to conserve
energy, respiring chemoorganoheterotrophic (coh) bacteria transfer
electrons from organic compounds to external electron acceptors that
are mostly also inorganic compounds. Fermenting organisms are also
chemoorganoheterotrophs that use biochemical reactions for a direct
formation of energy-rich bonds. Some phototrophs can grow pho-
tolithoautotrophically (pla), others photoheterotrophically (ph) or pho-
tomixotrophically (pm). Note that energy-rich bonds can also be used
to build an ion motive force, for example, for transport processes, and an
ion motive force to drive electrons toward a low redox potential (reverse
electron transport.)
occur as electron pair of one atom or as bonding electron
pair between atoms.
The state of electrons in an atom is described by
the Schrdinger equation. The mathematical solution
of this equation, which can be solved exactly for the
hydrogen atom and approximated for all other atoms,
gives three-dimensional residence probabilities (orbitals)
for a given electron or electron pair in the atomic shell.
To solve the Schrdinger equation, three dierent but
connected quantum numbers have to be assumed. The
principal quantum numbers (n = 1, 2, 3, 4, 5, ) roughly
refers to the energy of the electron. The higher n is, the
higher the energy of an electron and the farther away from
the nucleus it resides. The azimuthal quantum number
l describes the orbital angular moment, the geometrical
form of the residence probability, and is between 0 and
(n1). The magnetic quantum number m, nally, describes
S10 S1 Basic Biochemical Roots

Box S1.2: Why do electrons not fall into the atomic nucleus?

Why do electrons not fall into the atomic nucleus in
spite of the electromagnetic attraction between electrons
and protons? First, p, d, and f orbitals have a residence
probability of the electron in the nucleus of zero because
they have at least one axis of symmetry; the wave function
is zero at this position. The s orbitals have some residence
probability for their electrons in the nucleus; however,
as the nucleus is too small, this value is extremely low
for the boundaries of the nucleus. The only allowed
interaction of an electron with a proton here would be
the formation of a neutron. This reaction needs another
particle, an antineutrino. For most atoms the probability
of an electron in the nucleus combined with the presence
of an antineutrino is so small that their transformation
into another element (with one neutron more and one
proton less) cannot be measured; they are stable. Some
atoms, however, are unstable and perform a reaction
called electron capture. This form of a radioactive decay
releases gamma radiation resulting from the movement
of a 1s electron into the nucleus. An example is the iron
isotope 55-Fe that decays into 55-Mn with a half-life of 2.6
years. So, for the isotopes of some radioactive elements,
electrons do fall into the atomic nucleus from time to time
but, fortunately, the atoms of most elements are stable.

the magnetic moment of the electron and is between l
and l.
Taking this together, at n = 1, the K shell, l = 0, and
m = 0, meaning that only one orbital exists with no axis
of symmetry, the 1s orbital. The single electron in atomic
hydrogen resides here, 1s1 , and an electron pair in He,
indicated by 1s2 . The atomic K shell cannot take more
electrons so that the next shell with n = 2 has to be used
in case of Li, which contains 3 protons and thus needs 3
electrons in a neutral atom. The two electrons in the K shell
of He are in the same state with the exception of their spin
and are attracted by two protons. They are located much
closer to the nucleus than the single electron in atomic
H. This makes atoms with lled electron shells extremely
stable, low in energy, and happy, and that is why He is
a noble gas that does not need to share its electrons with
other atoms. This denes the rst period of the periodic
system of the elements (Figure S1.3).
Starting with Li, n = 2, and l = 0 or l = 1. Again, there
is a 2s orbital at l = 0 but three dierent orbitals at l = 1,
the three 2p orbitals, that have one symmetry axis each.
They are dened by m = {1, 0, 1} and named 2px , 2py , 2pz
orbitals because they are oriented along the three dierent
axis of a coordination system. Thus, the second or L shell
is able to accommodate an electron pair in the 2s orbital
(Li 2s1 , Be 2s2 ) and three electron pairs in the three 2p
orbitals, which are lled up from B via C, N, O, F to Ne
(1s2 2s2 2p6 ). Again, in Ne, a shell is completed and all

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18
s1 s2 s2
p1 p2 p3 p4 p5 p6
d1 d2 d3 d4 d5 d6 d7 d8 d9 d10
f0 f1 f2 f3 f4 f5 f6 f7 f8 f9 f10 f11 f12 f13 f14
1 1H 2He

2 3Li 4Be 5B 6C 7N 8O 9Fe 10Ne

3 11Na 12Mg 13Al 14Si 15P 16S 17Cl 18Ar

4 19K 20Ca 21Sc 22Ti 23V 24Cr 25Mn 26Fe 27Co 28Ni 29Cu 30Zn 31Ga 32Ge 33As 34Se 35Br 36Kr

5 37Rb 38Sr 39Y 40Zr 41Nb 42Mo 43Tc 44Ru 45Rh 46Pd 47Ag 48Cd 49In 50Sn 51Sb 53Te 53I 54Xe

6 55Cs 56Ba 57-71 72Hf 73Ta 74W 75Re 76Os 77Ir 78Pt 79Au 80Hg 81Tl 82Pb 83Bi 84Po 85At 86Rn

7 87Fr 88Ra 89-103 104Rf 105Db 106Sg 107Bh 108Hs 109Mt 110Ds 111Rg 112Cn 113Uut 114Fl 115Uup 116Lv 117Uus 118Uuo

57La 58Ce 59Pr 60Nd 61Pm 62Sm 63Eu 64Gd 65Tb 66Dy 67Ho 68Er 69Tm 70Yb 71Lu

89Ac 90Th 91Pa 92U 93Np 94Pu 95Am 96Cm 97Bk 98Cf 99Es 100Fm 101Md 102No 103Lr

Figure S1.3 Periodic system of the elements. The periods are
dened by the principal quantum number n of the valence elec-
trons. Groups 1 and 2, the alkali and earth alkali metals, ll up
their respective s orbitals and elements of the groups 1318 their
p orbitals. In-between are the transition metals, which ll up the d
orbitals. Not shown are the lanthanides (rare earth elements) and
actinides, which ll up f orbitals. No elements are known that ll
up the 5g or 6g orbitals. Such a process should start with elements
of period 8; however, the element with the highest atomic number
and highest atomic mass discovered so far is the articially synthe-
sized and very unstable ununoctium (118-Uuo) of group 18 and
period 7. Its half-life is below 1 millisecond.

electrons are exceptionally close to the atomic nucleus.
This denes the second period of the periodic system of the
elements, which contains three of the four most prominent
major bioelements, C, N, and O (Figure S1.3, see Section
S1.1.5).
Starting with Na, n = 3 allows l = {0, 1, 2} indicating that
this M shell contains a 3s orbital for one electron pair, three
3p orbitals for three electron pairs, and ve d orbitals with
m = {2, 1, 0, 1, 2}, which have two axis of symmetry
and can harbor ve electron pairs. At n = 4, f orbitals with
m = {3, 2, 1, 0, 1, 2, 3} with three axis of symmetry come
into play and with n = 5, theoretically, g orbitals. However,
starting with the 4s orbital, some orbitals with a higher
principal quantum number have a lower energy than d- or
f orbitals of shells with a lower principal quantum number,
and are lled up earlier. So, moving through the periodic
systems of the elements from one element to the next one
with increasing atomic number (Figure S1.3), the various
elements are lled up in the order s, s, ps, ps, dps, dps,
fdps, fdps: 1s (H, He); 2s (Li, Be); 2p (B-Ne); 3s (Na, Mg);
3d (Al-Ar); 4s (K, Ca); 3d (Sc-Zn); 4p (Ga-Kr); 5s (Rb, Sr);
4d (Y-Cd); 5p (In-Xe); 6s (Cs, Ba); 4f (La-Lu); 5d (Hf-Hg);
6p (Tl-Rn); 7s (Fr, Ra); 5f (Ac-Lr); 6d (Rf-Cn), and a few
articial elements with a very short half-life having elec-
trons in the 7p subshell. This rule holds true with very few
exceptions such as Cu that has 3d10 4s1 instead of 3d9 4s2 ,
because a lled 3d subshell represents a low state of energy.
From this simple consideration of the electron-lling
rules for the periodic system of the elements, a few impor-
tant conclusions can be drawn. First, there are groups of
elements that are similar in their arrangement of outermost
valence electrons. There is one single s1 electron in the
valence orbital in case of Li, Na, K, Rb, Cs, and Fr. These
elements behave similarly in chemical reactions and belong
to the same chemical group, the alkali metals or group 1. For
the same reason, other elements belong to chemical groups
such as group 14 comprising C, Si, Ge, Sn, and Pb or group
18, the noble gases. The elements of a chemical group dier
to some degree, however, because the valence electrons
are shielded in a dierent manner from the nucleus by
the other electrons, and they dier in energy because they
have dierent principal quantum numbers of their valence
electrons. Second, adjacent elements in the same period
also may be chemically similar, such as O and N. Thus, the
chemical features of an element are a simple consequence
of the Schrdinger equation and the number of electrons of
this element.
Note that a living cell uses only a small part of the periodic
system of the elements. Biologist can ignore the noble gases.
The 10 major bioelements are H in the rst period, C, N, O
in the second, P and S in the third, and four alkali and earth
alkali metals. The chemical elements up to selenium (atomic
number 34) contain most major and minor bioelements with
the exception of Mo, Cd in one exceptional example, I and
W (Figure S1.3).
S1.2 Energy and Transport S11
S1.2.3
Redox Energy and Electronegativity
Valence electrons that reside in an orbital of a shell that has
just started being lled up, such as the 2s1 electron of the
alkali metal Li, can lower their energy substantially when
they are being transferred to the last open position of the
shell of another element, such as F (1s2 2s2 2p5 ) in water.
In such a case, a Li+ cation is being formed that has the
same electronic conguration as He (1s2 ) and a F anion
that resembles Ne (1s2 2s2 2p6 ). That way, a couple of ions is
being formed and the previous valence electron of Li has a
much lower energy in F than it had in atomic Li, at least in
aqueous solution.
The individual conguration of the electron shell of atoms
arranges them into atoms that like to give their electrons
away, and to atoms that like to receive electrons. A conse-
quence of this is the standard redox or half cell potential of
elements in aqueous solution that increases from the earth
alkali metal Sr with a potential of Eo = 4.10 V and Li with
Eo = 3.04 V to F with Eo = +2.866 V. These values are nor-
malized to the potential of hydrogen that was dened to be
0 V at pH = 0. So, a low half cell potential indicates an elec-
tron donor, an element or compound that contains an elec-
tron or electron pair on a high state of energy. In contrast, an
electron acceptor has a high half-cell potential and loves
to receive an electron or electron pair.
The half cell potentials describe how much energy would
be released quantitatively if electrons are transferred, for
instance, from Li to F, Eo = 2.866 (3.04) V = 5.926 V at
1 M concentrations and pH = 0. The same calculation can
be done for any other couple of atoms and the reaction
proceeds always if Eo > 0. Using an augmented equation
(Nernst equation) also the real E value at concentrations
other than the standard conditions (1 M concentration,
pH = 0) can be calculated. Moreover, using the Faraday
equation G = nF . E (n, number of transferred elec-
trons, F, Faraday constant 96485 C/mol, charge of a mol of
electrons), the energy given in the unit V can also be cal-
culated into the unit J/mol. Because 1 J = 1 W s = 1 A V S
and 1 C = 1 A s, E = 1 V resembles about 100 kJ/mol
(96.485 kJ mol1 exactly) at 1 electron transferred.
So, the half-cell potential of a chemical element is a direct
consequence of the conguration of its electron shell. A
second consequence of this conguration is Linus Paulings
electronegativity, which increases from 0.7 for Fr via 1.0 for
Li to 4.0 for F. While the dierence in the half-cell potential
of two elements describes how much free energy is released
when a mol of electrons is transferred from one atom to
another, and the dierence in electronegativity can be used
to calculate if the electron is completely transferred to the
other atom (ionic bond) or is shared in the form of a covalent
bond between both, or anything in-between. Note that each
covalent bond between atoms with dierent electronega-
tivities means that this bond also has an ionic character: the
electron resides more frequently with the atom with higher
S12 S1 Basic Biochemical Roots
electronegativity than with that with lower electronegativ-
ity. This leads to partial charges in the molecule, a negative
partial charge for the atom with high electronegativity and
a positive partial charge for that with low electronegativity.
Partial charges of atoms in cellular molecules are at the very
core of all biochemical reactions inside a cell.
S1.2.4
Atoms in Molecules
In a covalent bond, a pair of electrons is shared between
two atoms. As outlined above, atomic orbitals are three-
dimensional wave functions that describe the residence
probabilities of an electron or electron pair around the
atomic nucleus. Consequently, molecule orbitals describe
the residence probabilities of the shared electron pair in
a covalent bond. These molecule orbitals originate by a
mixture of the atomic orbitals that contribute to the bind-
ing process; the atomic orbitals hybridize into molecule
orbitals. Only the outermost occupied s, p, and d orbitals of
the electron shell contribute to the molecule orbitals. As a
rule, the number of orbitals before and after the hybridiza-
tion process must be constant. So, if in the easiest example
the two 1s orbitals of atomic hydrogen hybridize to form
molecular hydrogen, two molecule orbitals are the result, a
binding and an antibinding * one. The binding orbital form four bonds in sp3 , or one and three bonds in
contains all the residence probabilities of the former atomic
1s orbitals that are located between the nuclei while the
antibiding * orbital possesses those that point away from
the binding partner. The two previous 1s electrons form an
electron pair with dierent spins that occupy the binding
orbital. As both electrons are now close to two nuclei each,
they can lower their energy and are more happy than
alone in atomic hydrogen. This leads to a decrease in the
energy level of the binding orbital compared to the former
atomic orbitals (although the spin of one electron has to be
inverted), while the energy level of the antibinding orbital
increases to the same extent. As a consequence, hydrogen
atoms form molecular hydrogen.
Considering helium, the atoms of this element contain a
pair of electrons in 1s. If two He atoms hybridize their 1s
orbitals into a binding and an antibinding molecule orbitals,
two of the resulting four electrons would occupy the bind-
ing orbital and the remaining two the antibinding orbitals.
The overall energy would be the same but the system would
contain one particle (a hypothetical He2 molecule) instead
of two, which is a decrease in entropy, and, consequently,
such a process is forbidden by the second law of thermody-
namics.
Elements with more valence electrons are able to form
up to three covalent bonds with a suitable partner atom in
a diatomic molecule. One of these bonds is always a bond
with the molecule orbital located along the binding axis.
The additional bonds are one or two molecule orbitals.
These orbitals have each two residence probabilities above
and below the binding axis, or in the case of a third bond,
additionally to the left and to the right of the axis. Of course,
if one s bond and two bond have to be formed, a total
of 6 atomic orbitals have to hybridize, and an antibinding
s* and two * molecule orbitals are also the result of the
hybridization. A remarkable insight coming from the
consideration of the electronic conguration of molecular
oxygen is given in Figure S1.4.
In addition to the orbitals of two dierent atoms, the dif-
ferent orbitals of a single atom may hybridize, provided they
are similar in size. This leads to the three possible structure
of a carbon atom in molecules: (i) sp3 , (ii) sp2 , or (iii) sp. In
sp3 all three 2p orbitals may hybridize with the 2s orbital
to form four 2sp3 orbitals, which point to the corner of a
tetraeder. These four sp3 orbitals hybridize again with the
atomic orbitals of four other atoms, for example, hydrogen
atoms in methane, leading to four orbitals. These are occu-
pied by an electron pair, one of the four valence electrons
from the carbon and one from the binding partner. In sp2
carbon, the pz orbital is left out from the hybridization and
is able to form a bond. The remaining three s bonds are in
the same plane and have an angle of 120 between them. In
sp carbon, the py orbital is additionally left out to form the
second bond while the two remaining s bonds line up with
an angle of 180 .
Thus, carbon has four valence electrons and may thus
sp2 , or two and two bonds in the sp state. Nitrogen
has one electron (and of course one proton) more, which
leads to the formation of a free electron pair in an sp3 , sp2 ,
or sp orbital. Such an orbital is now fully occupied and
does not engage in stable and covalent chemical bonds but
is very important for the formation of chemical bonds in
biochemistry. This leaves for nitrogen three bonds in sp3 ,
or one and two bonds in sp2 , or two and one bonds in
the sp state. Oxygen has yet a second electron more, leading
to two free electron pairs and the inability to form bonds in
the sp state because both sp orbitals would be occupied by
free electron pair; oxygen cannot form formal triple bonds.
This leaves for oxygen two bonds in sp3 , or one and one
bonds in sp2 . Fluorine adds a third electron, leading to
three free electron pairs and only the ability to form one
bonds in sp3 . Finally, Neon contains four free electron pairs
and does not form a chemical bond at all.
As the the chemical formula for the dry mass of living
matter can be summarized as <CH2 O> with O standing
for oxygen or nitrogen (see Section S1.1.5), the multitude
of dierent molecules in living cells is mainly constructed
of a very few basic components. Na+ , K+ , Mg2+ , and Ca2+
are metals that occur as mono- or divalent cations in the
cell. Phosphorus is nearly exclusively phosphate PO4 3 , sul-
fur predominantly present as sp3 sulfur with two free elec-
tron pairs and two s bonds, or as sulfate SO4 2 . Most of
the cellular components, however, are composed of C, H,
O, and N.
Combining this fact with Linus Paulings electronega-
tivity, the percentage ionic character of important bonds
 S1.2 Energy and Transport S13

Orbitals of the
3O -molecule
2

*p Orbital of an O-Atom
Orbital of an O-Atom
*y *z

2px 2py 2pz 2sp 2sp 2px 2py 2pz

Energy

y z

*s
2s 2s

Figure S1.4 Electronic conguration of molecular oxygen. The
panels to the left and to the right show the conguration of the 8
electrons in atomic oxygen that are in the 1s, the 2s, and the 2p
orbitals. The arrows represent the electrons and the direction of
the arrow the spin. Following Hunds rule and the higher energy of
a spin of 1/2, the two single electrons in 2p occupy one orbital
each. In the middle is molecular oxygen. The electrons in 1s do
not take part in the binding process and remain in atomic orbitals.
More precisely, they form a binding and an antibinding molecule
orbital. The same is true for the 2s orbitals but because of the
presence of the antibinding orbitals that point away from the bind-
ing partner, they hybridize rst with the 2px orbital in the same
atom yielding two 2sp orbitals. One 2sp orbital per atom subse-
quently forms a and an anti-binding * orbital. The remaining
two 2p orbitals per atom hybridize to two binding and two
antibinding * orbitals. Now, if all 16 electrons of molecular oxy-
gen are distributed, 4 reside in the 2 atomic 1s orbitals, another
4 in the 2 atomic sp orbital that points away from the respective
binding partner, 2 are in the binding orbital and 4 in the two
binding orbitals above and to the side of the binding axis. This
leaves two electron that occupy the antibinding * orbitals, again
both according to Hunds rule. Therefore, there is not a double
bond between the two atoms in molecular oxygen but a triple
bond plus two half antibonds. Second, oxygen is in the triplett
state and contains two single electrons that mediate a radical char-
acter of the molecule. Third, to perform a nonradical reaction, rst
the spin of one of the two electrons in the * orbitals has to be
turned to allow an electron pair in the other * orbital, leading to
singulett oxygen. This inversion needs energy. Because of the high
activation energy of molecular oxygen, the molecule is relatively
lazy in starting a nonradical chemical reaction. Would this not be
true, the complete biomass on the planet would burn to carbon
dioxide immediately. What a remarkable insight coming from a sim-
ple consideration of molecule orbitals!

Table S1.5 Ionic character of bonds between S1.2.5

macrobioelements. Functional Groups and Energy-Rich Bonds

Binding partner C (2.5) N (3.0) O (3.5) S (2.5) P (2.1)

So, the atoms that mainly form the dry biomass are C and N
(%) (%) (%) (%) (%)
in three possible electronic congurations, O in two, H in a
simple s bond, P as phosphate, and S mainly as thiol group
C (2.5) 0 6 22 0 4
SH. Because triple bonds are rare, exceptionally occurring,
H (2.1) 4 19 39 4 0 for instance, in molecular nitrogen and hydrogen cyanide
O (3.5) 22 6 0 22 39 HCN, this leaves an even shorter list of atomic constituents
of macromolecules and metabolites.
N (3.0) 6 0 6 6 19
The carbonyl group C=O contains C and O in the sp2
Mg (1.2) 34 55 74 34 19 conguration. Because of this, both atoms are in a plane
with the electrons below and above this plane. Because of
The percentage ionic character as calculated from the dierence in
the partial ionic character of the bond, the oxygen carries a
Paulings electronegativity (in parenthesizes) is given. Mg is the
macrobioelement with the highest electronegativity. partial negative charge and the carbon a positive one. This
leaves the nucleus of the carbon unprotected from above or
between the basic components of biomolecules can be below the plane for advancing free electron pairs coming
calculated (Table S1.5). The bonds CO, OH, SO as from a direction opposite to that of the oxygen. Such a free
in sulfate, PO as in phosphate, and NH are 22%, 39%, electron pair can come from a hydride (H ) component
22%, 39%, and 19% ionic, respectively, leading to a partial to reduce the carbonyl group to an alcohol group COH,
negative charge at the oxygen or nitrogen atom and a from a CH acidic bond leading to the formation of a
positive partial charge at the carbon, hydrogen, sulfur, or new CCOH arrangement, to the addition of water to
phosphorus atom. In contrast, the bonds CH, CS, CN, form an acetal HOCOH, to the addition of an alcohol
and CC have predominantly a nonionic character. group ROH leading to ROCOH, or a thiol leading to
S14 S1 Basic Biochemical Roots
RSCOH. Second, the oxygen of a carbonyl group with
its partial negative charge may steal a proton from an
adjacent CH group, which yields by proton migration in a
keto-enol-tautomerism an enol CHC=O > C=COH.
The ability of proton migration in a CH group adjacent
to a carbonyl group indicates that such a group is CH
acidic, meaning that the group may release under certain
circumstances (e.g., within the reaction center of enzymes)
a proton H+ . This reaction results in an intermediary car-
banion with a free electron pair, which is free to attack the
carbon of another carbonyl compound. Carbonyl groups
are named keto groups when they are in the middle of a
molecule and aldehyde group are terminal. Independent
of the position, carbonyl groups are the most important
functional groups in cellular metabolites.
The carboxyl group COOH is formally a terminal
carbonyl plus an alcohol group; however, both oxygen
atoms share the proton bound to the group and release
it with a pK a value (the pH value mediating a 50:50 ratio
between protonated and deprotonated form) usually
below 5. That means that carboxyl groups are com-
pletely deprotonated at a neutral pH value such as that
in the cytoplasm of most bacteria. The resulting nega-
tive charge is delocalized across all three atoms, which
represents a low state of energy. Amides (CONH2 )
and esters (COOR) are derivatives of carboxyl groups
with amines or alcohols, respectively, which represent a
medium level of energy (free energy of hydrolysis about
20 kJ mol1 ). The peptide bond (CONHC) is a
special form of an amide that connects amino acids in
proteins. The nitrogen atom is also in a sp2 state, allowing
the electrons to visit all three atoms, C, O, and N. This
leads to the stability of the peptide bond against rotation
and to a free hydrolysis energy lower than that of other
amides.
Acid anhydrides (COOOC) and thioesters (COSR)
are energy-rich derivatives of carboxyl groups (free energy
of hydrolysis of about 50 kJ ml1 under physiological
conditions). Because the phosphate, sulfate anions, and
the carboxyl group share a common structural motive,
a carbonyl-like structure plus an alcohol (O=POH in
H3 PO4 equaling O=P(OH)3 and O=SOH similarly in
H2 SO4 ), they can form mixed acid anhydrites in the
form COOOP/S, which are very important groups in
metabolites, for instance, attached to the C1 atom in 1,3
bis-phosphoglycerate (note that the C3 carbon contains
a phosphate ester and not a mixed acid anhydride). Two
phosphate anions are also able to form an acid anhydrite in a
POOOP polyphosphate bond such as in polyphosphate
or adenosine-5 -triphosphate between the / and /
phosphate groups. Finally, sulfate and phosphate also may
form a mixed acid anhydrite SOOOP as in adenosine-
5 -phosphosulfate APS, which is the rst metabolite in the
pathway of sulfate reduction.
In alcohol (COH) and amine (CNH) groups the
carbon is usually in the sp3 state. The N or O in either group
interacts with the solvent water because the hydrogen atom
carries a partial positive charge and is attacked by the free
electron pairs of the water oxygen and vice versa: the free
electrons of the partially negative N and O atoms attack
hydrogen atoms of the water molecules. Because of this
ability to form hydrogen bonds with the solvent water, the
presence of an alcohol or amine group leads to an increase
in water solubility of a metabolite. In contrast, methyl
(CH3 ) or methylene (CH2 ) groups are not able to
interact with water because the hydrogen atom does not
carry a partial positive charge. These groups decrease the
water solubility of a compound. If many of them are present
in a row, the respective molecule becomes expelled from
the water phase, is hydrophobic, and forms another phase
that is stabilized by hydrophobic interactions. Examples
are the lipid parts of phospholipids in a biological mem-
brane and hydrophobic regions of proteins that serve as
hydrophobic cores of a protein or embed it into a biological
membrane.
So, how are these functional groups arranged in the
four classes of macromolecules? Proteins are composed
of 20 classic amino acids plus some unorthodox ones
such as selenocysteine in a peptide bond. Amino acids
are composed of a carboxyl group followed by an amine
group in the optical L-conformation and a functional tail,
which may be simply a hydrogen or a CC chain with
an alcohol, thiol, carboxyl, amide, amine group, aromatic
ring, or hydrophobic region, respectively, depending on the
individual amino acid. The core of biological membranes
in most organisms is composed of phospholipids that
contain glycerol (three alcohol groups in a row), phosphate
in an ester bond to glycerol, additional groups attached to
the phosphate, and two fatty acids in ester bonds to the
other two alcohol groups of the glycerol. Fatty acids are a
carboxyl group followed by many methylene and a terminal
methyl group, which makes the molecule hydrophobic
(e.g., palmitic acid is COOH(CH2 )14 CH3 ). Some fatty
acids may contain one or more double bonds between
two adjacent carbon atoms or a branching methyl group.
Carbohydrates are composed of monosaccharides with
the sum formula (CH2 O)n , which is that of the biological
dry mass in general. They are mainly a chain of alcohol
groups. As the terminal alcohol group needs an additional
hydrogen atom, one of the other carbons of the molecule
must carry a carbonyl group to keep the sum formula. In
ketoses this is mostly the C2 atom and in aldoses the C1
atom. Derivatives of monosaccharides are amino sugars
with an amino group instead of an alcohol group, sugar
acids with a carboxyl group or sugar alcohol with the car-
bonyl group being reduced to an alcohol group. In nucleic
acids, nally, the building blocks are a sugar (ribose in RNA
or 2 -desoxyribose in DNA) carrying a phosphate group at
the C5 atom in an ester bond and a purine or pyrimidine
base at C1. So, the macromolecules can be easily assembled
from C, H, O, N, S, and P, which are predominantly present
in the form of a few building blocks.
 S1.2 Energy and Transport S15

S1.2.6 Table S1.6 Photon wavelengths and energies.

Energy Sources for Life: Light Energy
Name of the radiation Wavelength Energies (mol)
S1.2.6.1 How to Harvest Photons
If a cell harvests light energy or physical energy, photons Gamma rays <10 pm >12 GJ
coming from the sun have to be absorbed and transformed
X-rays 10 nm100 pm 12 MJ1.2 GJ
into usable energy forms. Photons are light particles and
light is the same time a light wave. Photons migrate with Far to extreme UV 200 nm10 nm 598 kJ12 MJ
the speed of light (c, about 300 000 km s1 ) in vacuum, UV_C 280 nm200 nm 427 kJ598 kJ
and the energy of a single photon depends solely on its
UV_B 315 nm280 nm 380 kJ427 kJ
wavelength following the famous equation E = hc/,
with h being the Planck constant (6.626 1034 J s1 , UV_A 400 nm315 nm 300 kJ380 kJ
hc = 198.7 1027 J m1 ). Thus, an Einstein (mol of pho- Blue light 490 nm400 nm 244 kJ300 kJ
tons) has an energy content of 0.1197/ J m mol1 , resulting
Green light 540 nm490 nm 222 kJ244 kJ
in about 120 kJ mol1 at a wavelength of 1 m = 1000 nm
and 1200 kJ mol1 at 100 nm. Yellow light 570 nm540 nm 210 kJ222 kJ
As photons are the exchange particles of the electromag- Orange light 630 nm570 nm 190 kJ210 kJ
netic force, harvest of photons can be done only by using an Red light 740 nm630 nm 162 kJ190 kJ
electromagnetic interaction. This always has something to
do with electrons, which either constitute the electron shell IR_A 1.4 m740 nm 85.5 kJ162 kJ
around an atoms nucleus, are shared between atoms to form IR_B 3 m1.4 m 40 kJ85.5 kJ
a chemical bond, or migrate freely within solid bodies in IR_C 1 mm3 m 120 J40 kJ
metals or semiconductors. Dierent amounts of energy, and,
Far infrared 15 mm1 mm 8 J120 J
therefore, dierent photon wavelengths, are needed to inu-
ence these processes. To remove the rst electron from an Microwaves 1 m1 mm 120 mJ8 J
atom needs between +378 kJ/(Cs) and +2,381 kJ/mol (He), Radio waves, THF to UHFa) 1 m1 mm 120 mJ8 J
with the energy increasing for the second, third, or what-
Radio waves, VHF to LFb) 10 km1 m 12 J120 mJ
ever electron, for example, +12 000 kJ mol1 for the seventh
electron of Ar. For these processes, photons must have a Other radio waves >10 km <1.2 J
wavelength of 316 nm (Cs), 50 nm (He), or 10 nm (seventh of
a) Used for GPS and mobile phone communication.
Ar), respectively. Therefore, photons with wavelength below
b) Used for broadcasting.
300 nm (for most atoms below 200 nm) are constituents of
ionizing radiation in the form of high-frequency ultravi-
olet light, X-rays or gamma rays. These photons cannot character of the compound is obtained. This problem can
be harvested because they damage the cellular components be solved by conjugated double bonds. In these, a pair of
(Table S1.6). double bonds is separated by an intervening single bond.
Likewise, the energy of photon with a wavelength of This allows the electrons of both double bonds to move
197, 111, and 354 nm is suitable to split the most frequent across all two double bonds and across the single bond,
bonds in cellular macromolecules, the single covalent bonds and the electrons become delocalized electrons. If a
between CC, CO, and CH, respectively, corresponding photon is absorbed, an electron moves from the binding
to +607, +1076, and +338 kJ mol1 . Again, this is the range molecule orbital with the highest energy to the antibinding
of UV light. When photons of this frequency split single or nonbinding molecule orbital with the lowest energy. The
covalent bonds, one electron is left at each of the previous respective dierence in energy is termed frontier orbital
binding partners. Such atoms are called radicals. Radicals gap. As the electron in the antibinding orbital is also able
are atoms of molecule groups that tend to react quickly to move across the complete conjugated system, the radical
with other compounds. A few biochemical reactions use character is smeared across the molecule, which decreases
radicals; however, in these reactions, the generation of the the reactivity of the excited molecule.
radicals and the reaction pathway is tightly controlled. UV A system of two conjugated double bonds absorbs UV
light would generate radicals in an uncontrolled manner light and represents a dangerous, excited high energy
that might damage cellular components. Thus, photons state; however, more than two double bonds can also
that have the energy to destroy single covalent or bonds become conjugated double bonds if the single double
cannot be harvested. bonds are always separated by exactly one bond. At
This leaves electrons in double or bonds as receptors more than eight conjugated double bonds, photons of
for photons that can be harvested. However, absorption of the visible light are able to push electrons across the
a photon gets the electrons also from a binding into an frontier orbital gap and the resulting excited state is less
antibinding (*) molecular orbital, so that again a radical dangerous than that of a single or bonds. The more
S16 S1 Basic Biochemical Roots
double bonds are conjugated the smaller is the photon
energy needed to bridge this gap. An example for such a
molecule is -carotene that contains 11 conjugated double
bonds and is colored orange. Alternatively, a metal cation
can be hooked up with a conjugated system in a charge
transfer complex, which decreases the frontier orbital gap
even further. Here, chlorophyll may serve as an example,
which contains a magnesium cation in its center and
10 or more conjugated double bonds, depending on the
individual chlorophyll compound. Bacteriochlorophyll b
is able to absorb photons up to a wavelength of 1040 nm
(115 kJ mol1 ), which is already in the infrared region. It
may be dicult to harvest photons with even higher wave-
lengths, because at about 2 m photons start to stimulate
molecule vibrations.
Thus, by using molecules that contain conjugated double
bonds and/or charge transfer complexes, photons of the
visible light can be harvested. Note that the visible (plus
near IR and UV) light is the most useful light for biolog-
ical processes because light of shorter wavelength is too
dangerous to be used and light with a longer wavelength
just stimulates vibrations. To use these photons, they have
to be absorbed, which leaves the nonharvested photons
to be reected and scattered, resulting in a color of the
light-absorbing structure.
Absorption of the photons of visible and near IR light
leads to an electron per molecule that has moved across the
frontier orbital gap and resides in an antibinding * orbital.
Such an excited state is called an exciton. An exciton is not
stable and is quenched within fs (femtoseconds, 1015 s),
which means the electron falls back across the gap and
the released energy usually stimulates thermic molecular
vibrations. Carotinoids and other compounds may be
used to quench unwanted excitons before they can cause
any harm.
On the other hand, phototrophic (light-eating)
organisms harvest light in large light-harvesting com-
plexes composed of proteins, chlorophyll, or another
chromophor such as phycobilins. These light-harvesting
complexes contain their respective chromophores in
a highly ordered arrangement such as 18 chlorophyll
molecules in a ring with the individual molecules in an 90
angle to the plane. This allows stabilization of the excitons
by quantum entanglement until the exciton energy can
be used. To accomplish this, the exciton is transferred
into a photosynthetic reaction center adjacent to the
light-harvesting complex, more specically to a pair of
chlorophyll molecules, the special pair. From here, not the
exciton state but an excited electron jumps within 0.2 ns to
an adjacent phaeophytin molecule, which is a chlorophyll
without magnesium, that is, without the contribution
of a charge-transfer complex. Thus, phaeophytin has a
much larger frontier orbital gap than chlorophyll and can
store an additional electron without the danger of rapid
quenching. The trick of this reaction is that light energy
is used to transfer an electron with a low energy (positive
redox potential) into an electron with high energy (lower
or even negative redox potential). At this stage, the energy
of the photon has been partially conserved by creating a
compound with a low redox potential.
S1.2.6.2 How to Conserve Light Energy as Redox Energy
Migration of electrons from a low to a high redox poten-
tial (E = Ea Ed ; with E being the half cell potentials
of the electron acceptor and donor) releases free energy
(G = n F E) that can be transformed into other forms of
energy. In the light-harvesting reaction centers, an exciton
was transferred from the light-harvesting complex to the
special pair of chlorophyll molecules in the reaction center,
and from here an electron to an adjacent phaeophytin
within 0.2 ns. Within further 200 s, the electron is further
transferred to another electron carrier, a ubiquinone, which
usually have standard redox potentials at pH = 7 of about
0 mV. At the same time, the special pair is reimbursed
with an electron from a second electron carrier in this
photosynthetic primary reaction. It should be noted
that another type of photosynthetic reaction center uses
another electron acceptor (iron-sulfur centers) instead of
ubiquinone.
S1.2.7
Hierarchy of Transport Processes
Any dierence in the concentration of a component is
a form of energy because as a consequence of entropy,
components have to be distributed in as much space as
possible. The dierence in the energy of two concentrations
of a component can be calculated as G = RTln K (R,
general gas constant 8.3131 J mol1 K; T, absolute tempera-
ture in K; ln, natural logarithm to basis e, the Euler number;
and K, the quotient of the two concentrations). This is the
most important process for any chemical reaction that is
usually not running at 1 M concentrations. The free Gibbs
energy G yielded by the reaction is the sum of the free
Gibbs energy at 1 M concentrations plus the concentration
terms for substrates and products. On the other hand, if
the substrate and product terms for the concentrations
are equal to the free Gibbs energy for 1 M concentrations,
the reaction stops because the overall G = 0. In this
case, a thermodynamic equilibrium is reached. Because
of the constant demands of energy transformation, living
cells never reach a thermodynamic equilibrium of their
biochemical processes (see Section S1.1).
The term G = RTln K is also true to describe con-
centration dierences in dierent compartments, such as
outside and inside a cell or in two dierent cellular com-
partments. Because of entropy again, molecules want to
equalize this dierence in concentration and thus to reach
the thermodynamic equilibrium. They are, therefore, driven
to migrate from a compartment of high concentration to one
with lower concentration. If only concentration dierence
drives this migration process, it is termed diusion.
 S1.2 Energy and Transport S17

Diusion may occur unaided between dierent positions acceptor drives the transport event. Res-
in one compartment. The rate of diusion can be calculated piratory electron transport chain.
by Ficks rst law vdi = dc/dt = D A/V .(dc/dx): The rate c. Driven by a chemical reaction such
of the change in concentration vdi = dc/dt depends on as ATP-hydrolysis. Families of trans-
(i) the diusion coecient D; the area A across which port ATPases (ABC, P-type, F1 F0 ),
the components diuse; (iii) the volume V ; and (iv) the gradient-forming decarboxylases.
decrease of the concentration dc/dx. Diusion is rather 2. Secondary transport. Active transport driven
fast in gas, 10 times slower in liquids, and even slower by a concentration gradient, therefore
in solid substances. Examples are diusion coecients gradient-using transport.
of molecular hydrogen in air at 20 C of 108 m2 s1 , urea a. Uniport: a charge gradient drives the
in water at 20 C of 1.27 109 m2 s1 , and zinc in lead at transport reaction. Only the substrate
285 C of 7.5 1011 m2 s1 . Cells, however, are very small. It is transported propelled by its charge.
takes for a urea molecule, for instance, half a millisecond to Uptake of Mg2+ by CorA-like transporters,
reach the other cell pole, and a protein in a real experiment export of arsenite by ArsB- and chromate
is determined to be only 141 times slower than urea. by ChrA-like transporters.
Second, diusion may happen across a biological b. Symport: the substrate is transported
membrane. Migration of a molecule across a biological into the same direction as a second ion
membrane is termed membrane transport. Small molecules or molecule, which forms the driving
such as molecular hydrogen or ethanol may diuse freely concentration gradient. Lactose:proton
across a biological membrane. In this case the transport symporter LacY .
process is called simple diusion (I, Figure S1.5). c. Antiport: the substrate is transported
into the opposite direction of the
I. Simple diusion. Driven only by a concentration
second ion or molecule, which forms
gradient. Transport rate depends on the concentra-
the driving concentration gradient.
tion dierence according to Ficks Law. Transport of
Sodium:proton-antiporter NhaA.
molecular hydrogen and oxygen across the cytoplasmic
membrane.
II. Carrier-mediated transport. Catalyzed by a carrier,
mostly a trans-membrane protein. Faster than simple
Hydrophobic/ weakly polar Passive
diusion but showing substrate-saturation. S
compounds/ xenobiotics, gases
S
diffusion
Passive
A. Facilitated diusion. Carrier-mediated transport transport
driven exclusively by the concentration gradient of Channels Facilitated
S (Pores) S transport
the substrate. No accumulation. Transport through
outer membrane porins, glucose and glycerol facil-
itators.
B. Active transport. Carrier-mediated transport
driven by energy dierent from the concentration ATP ABC proteins
gradient of the substrate. Accumulation possible S
(e.g. P-gp, MRP)
S
ADP
on either side of the membrane, which may lead Primary
to concentration gradients. +
ATP +
Na ATPases Na
1. Primary transport. Active transport driven by K+ K+
an energy form that is not another gradient, ADP

therefore a gradient-forming transport.

Active ATP
a. Driven by light energy: Photons are X Certain SLC transporters, X
transport S prokaryotic transporters S
harvested as exitons. Their energy is used ADP
Secondary
to change the conformation of a trans-
Certain SLC transporters,
porter and the resulting conformational S+ prokaryotic transporters S+
tension is relaxed by a transport event.
Alternatively, the redox potential of a ATP Certain SLC transporters,
H+ H+
compound is decreased by the exiton S prokaryotic transporters S
energy, leading to electron transport, ADP

consecutively resulting in a transport

event. Bacteriorhodopsin, photosynthetic
light reaction.
b. Driven by redox energy: electrons trans- Figure S1.5 Hierarchy of transport processes (top) and examples
fer from an electron donor to an electron (bottom).
S18 S1 Basic Biochemical Roots
Biological membranes have a hydrophobic core. Most
molecules in the cytoplasm or molecules that cells would
like to have in the cytoplasm, however, are hydrophilic
substances, even charged ones, or large molecules up to
macromolecules. For such substances the diusion coef-
cient for transport across a biological membrane would
be extremely small. To deal with this problem, cells have
carriers that mediate (II, Figure S1.5) carrier-mediated
transport. Such carriers can be small compounds that
bind a substrate molecule on one side of the membrane,
migrate as carriersubstrate complex across the mem-
brane, and release the substrate on the other side of the
membrane. In most cases, however, such carriers are
integral membrane proteins that span and bridge the
biological membrane, allowing the substrate molecule
to travel through the interior of the protein to the other
side. All forms of carrier-mediated transport systems
show substrate-saturation: the rate of transport increases
initially linearly with increasing substrate concentration. At
higher substrate concentrations, the rate increases only a
little bit even when the substrate concentration increases
further and further. This is because only a limited number
of carriers is available to mediate the transport process and
this process takes time. When all available carriers mediate
transport with their highest possible turnover number, the
maximum transport velocity vmax is reached, which is the
product of the turnover number and the concentration of
the carrier. The rate v for most transport reaction follows
the MichaelisMenthen equation v = vmax cs /(K m + cs ) like
enzymes. Here, cs is the substrate concentration and K m
the MichaelisMenthen constant. It is easy to see that at
cs = K m the transport rate reaches half of vmax . Moreover,
at cs K m , v = vmax cs /K m (linear increase with the slope
vmax /K m ) and at cs K m , v = vmax .
If no other energy than the concentration gradient is used
to drive the transport process, this form of carrier-mediated
transport is called (IIA, Figure S1.5) facilitated diusion. If
facilitated diusion is allowed to take its time, this leads to
the same concentration of the substrate on either side of
the membrane, to a thermodynamic equilibrium. In con-
trast, in case of (IIB, Figure S1.5) active transport, other
energy forms are used to drive the transport reaction. In
this case, the other energy form is transformed into trans-
port energy, meaning that a substrate can be accumulated
on either side of the membrane. The formula to describe this
process is again G = RT ln K. In this case, an energy of
+5.8 kJ mol1 is needed to accumulate a substance 10-fold,
+11.6 kJ mol1 100-fold, +17.4 kJ mol1 1000-fold, and so on
(T = 303 K or 30 C).
In primary active transport systems (IIB1, Figure S1.5)
various forms of energy can be used to drive the active
transport process but not the concentration energy in
the gradients of other substances. These forms of energy
can be light energy (IIB1a), redox energy (IIB1b), or
the energy stored in energy-rich bonds of bioorganic
molecules (IIB1c). There are several examples for chemical
reactions that drive a primary active transport of the
category IIB1c, such as a decarboxylation reaction and
reduction of an organic heterodisulde; however, most
reactions of this kind are driven by a dephosphorylation
reaction.
In secondary active transport (IIB2, Figure S1.5), the
concentration energy of one substance is used to form a
concentration gradient of a second substance. The reaction
is called symport if both substances are transported into the
same direction (IIB2a) and antiport if they are transported
into an opposite direction (IIB2b). Uniport (IIB2c) is very
often confused with facilitated diusion. In case of uniport,
a charge gradient drives the formation of a concentration
gradient and uniport is, therefore, active transport. In
contrast and as stated above, facilitated diusion is driven
only by the concentration dierence of one substance, the
substrate, at either side of the membrane.
S1.2.8
Ion Motive Forces
When ions have dierent concentrations on both sides of a
biological membrane, these gradients contain two forms of
energy, concentration energy (G = RT ln K), and charge
(electrostatic) energy that results from the separation
of a positive charge on one side of the membrane from a
negative charge on the other side. A biological membrane
is an insulator in this aspect and functions as an electrical
capacitor. Using the Faraday equation (G = nFE), the
concentration energy can be calculated in the dimension V
of an electric tension or voltage: R T ln K = n F E and,
therefore, E = R T/(n F)ln K. In total, the energy of an ion
gradient is = + RT/(n F)ln K.
Many ions may form gradients across a biological
membrane but only a few of these gradients are used to
drive secondary active transport or other processes.
In such a case, the respective gradient is called an ion
motive force. Examples are Na+ ions and most important
protons, H+ , which build a sodium motive force (Na+ )
or a proton motive force (the pmf H+ ), respectively.
As the pH value indicates the proton concentration
with pH = lg [H+ ], [H+ ] = 10pH , K = 10pHi /10pHo
= 10pH , and ln K = ln(10pH ) = pH ln10. This leads
to H+ = + RT/(nF)ln K = + RT/(n F) ln10;
pH = + ZpH with Z = RT/(n F). ln10, which is 60
mV at 30 C. In bacteria, the pmf is about 200 mV with
positive charges outside, which calculates to an energy of
about 20 kJ mol1 that is released when one proton is
imported back inside. The dierent parts of the pmf,
and Z . pH, may contribute dierently to the overall pmf,
and this depends on the pH value of the growth medium
outside the cells and the internal pH value, which is 7.6 in
many bacteria. So, if the pH value of the growth medium
is neutral (pH = 7), pH = 0.6 and ZpH = 36 mV. The
remaining 164 mV of a pmf of 200 mV has to be contributed

by . In any case, the positive charges are outside of the
bacterial cell.
Compared to the pmf, sodium motive forces occur less
frequently, mostly as gradient additional to the pmf, or in
organisms living at high temperature of pH values. The
bacterium Escherichia coli (Bacteria, Gammaproteobacte-
ria) controls its internal pH value in a short-term response
by a sodium-proton antiporter. If the internal pH value
becomes too high, enhanced import of protons neutralizes
this value again, and forms a sodium gradient, which can
be used for sodium-driven transport processes. Vibration
of the components of biological membranes increases with
increasing temperature until protons of the pmf start to
diuse through the membrane and the pmf collapses. This
problem can be solved by using other components to a
certain point, but at the end, all start to leak protons. The
solution is to use sodium ions that are much bigger than
protons. Finally, at high pH values outside the cell, Z . pH
becomes negative (e.g., 180 mV at an outside pH of 10.6
and an internal pH value of 7.6). If the portion of the
pmf is no longer able to compensate this negative Z pH,
the solution could be again to use a sodium motive force
instead of a pmf.
S1.2.9
How to Built up a Proton Motive Force by Redox Energy
Description of the harvest of protons has been outlined
to the point that electrons have been transferred to an
ubiquinone. This redox energy between the chinone and
the electron donor of the photosynthetic reaction center
can be used to export a proton against a pmf of 200 mV. A
note of caution: redox energy and the energy in ion motive
forces is noted in the dimension V; however, both kinds
of energy are clearly dierent. Nevertheless, both forms of
energy can be transformed into each other.
Chinons such as ubichinon and menadione are important
constituents of electron transport chains in many organ-
isms. Both compounds dier somewhat in the exact value
of Eo but they share the same feature: they are reduced by
the transfer of two hydrogen moieties (a pair of electrons
and a pair of protons) to these compounds, and they are
hydrophobic constituents of biological membranes. In
contrast to other components, chinons are even able to
form stable radicals after transfer of only one electron and
proton to the chinon. So, they can occur as reduced form,
oxidized form, and radical intermediate. Cytochrome c-like
compounds contain a heme group with a single iron atom
in its center. They can store and transfer one electron but
no protons.
In general, there are three mechanisms that allow to build
up a pmf by using redox energy: (i) proton pumps, (ii) cou-
pling of a proton-releasing chemical reaction outside the cell
with a proton-consuming reaction inside, and (iii) a chinon
or Q cycle.
S1.2 Energy and Transport S19
H+ H+ H+
IV Cytc
III Q
I
SDH II
H+ H+
H2O O2 Fumarate H+
Succinate NADH
NAD++H+
TCA
Figure S1.6 The respiratory chain of mitochondria. This arrange-
ment of membrane-bound electron-transferring protein complexes is
similar in mitochondria and many strictly aerobic bacteria. The upper
side of this scheme represents the mitochondria matrix or bacterial
cytoplasm, the lower side the mitochondrial intermembrane space or
bacterial periplasm. Electrons mostly from NADH are transferred in
complex I via avin groups and ironsulfur centers to chinons and this
downhill movement of the electrons is coupled to the export of pro-
tons to build up a proton motive force. In complex III, the electrons are
further moved to cytochrome c and complex IV from here to molecu-
lar oxygen, forming water. Both transfers are also coupled with proton
export. In addition, electrons from succinate are transferred in complex
II to chinons without proton transport. All protons exported are used
on the way back to drive ATP synthesis in the F1 F0 -ATPase, which is not
shown here. (Graphics: D. Dobritzsch.)
Proton pumps are mechanoproteins that transfer elec-
trons from an electron donor to an electron acceptor. The
distance between the binding sites of either compound
is bridged by electron-transferring prosthetic groups: (i)
avine compounds, avin-adenine-dinucleotide (FAD) or
avin mononucleotide (FMN), which are able to separate
a hydride (H ) group into a proton (H+ ) and two electrons;
(ii) iron-sulfur clusters, mostly [2Fe-2S] or [4Fe-4S], which
transfer one electron; (iii) tightly bound chinon units that
may stabilize an intermediate radical; (iv) heme groups
that transfer single electrons but at a more positive redox
potential than iron-sulfur cluster; and (v) copper ions.
These electron-transferring prosthetic groups are arranged
in a distance of about 10 (1 nm) apart from each other.
While electrons are allowed to tunnel such a distance,
the bigger protons are not. Electron transfer from a donor
with a low to an acceptor with a high redox potential
via these prosthetic groups transfers the energy that is
released by each transfer step into conformational energy,
which is subsequently released by the transfer of a proton
against the pmf. Proton pumps may also catalyze the
inverse reaction: import of a proton drives a conformational
change and the resulting conformational energy is used
to move electrons into the opposite direction from a
donor with a positive redox potential to an acceptor with
a smaller potential. This process is called reverse electron
transport.
S20 S1 Basic Biochemical Roots
The two most important proton pumps are the NADH
oxidase and the cytochrome c oxidase. The NADH oxidase
(complex I in mitochondria, Figure S1.6) accepts electrons
from the cofactor NADH that transfers hydride ions H- at
a potential of Eo = 320 mV to chinons. The prosthetic c reductase at the inner face. At this site, the chinon
groups in the NADH oxidase are a avine and iron-sulfur
centers. Electron transfer drives export of three protons
against the pmf; an electron pair transported releases
640 mV, and 600 mV are needed for export of three
protons, so only 40 mV are used for entropy. The parts of
the protein that transfer electrons is separated from three
parts that comprise proton pathways, one for each proton
transferred per electron pair, so that proton and electron
transport can be coupled only by conformational energy.
The low amount of heat released and the separation of
both transport functions may be a prerequisite to allow
reverse electron transport. Some bacteria, however, contain
a nonpumping NADH-oxidase, sometimes in addition to a
proton-pumping one. Such a nonpumping oxidase cannot
be used for reverse electron transport.
The second important proton pump is the cytochrome
c oxidase (complex IV, Figure S1.6). This protein complex
transfers electrons from cytochrome c via copper atoms and
heme sites to molecular oxygen, pumping out two protons
per electron pair. Likely, both processes are again coupled
via intermediate conformational energy.
The easiest way to generate a proton motive force is to
consume protons by a chemical reaction inside the cell
and release protons outside. Two examples may illustrate
this process. Lactic acid bacteria (Bacteria, Firmicutes) live
by fermentation of carbohydrates into lactic acid. When
this compound is exported, it deprotonates outside of
the cell immediately into lactate and a proton. Second,
some bacteria that respire with sulfate as electron acceptor
produce molecular hydrogen in the cytoplasm using an
enzyme called hydrogenase. These bacteria possess a second
hydrogenase attached to their cytoplasmic membrane but
pointing to the outside. Production of molecular hydrogen
in the cytoplasm consumes electrons, the hydrogen diuses
to the outside, and is here assimilated again by the second
hydrogenase. The electrons are ultimately transferred to the
sulfate (more precisely APS or sulte) while the protons are
released.
The most complicated in generating a proton-motive
force is a special case of consumption inside and release out-
side, the Q cycle. This process is responsible for 50% of the
energy conservation in aerobic respirations and, therefore,
very important. Q cycles are performed by protein com-
plexes such as the cytochrome c reductase (complex III of
mitochondria, Figure S1.6). This protein complex contains
two binding sites for the electron donor ubichinon, one at
the outside face of the cytoplasmic membrane, one at the
inside face. A chain of cytochrome b prosthetic groups con-
nect both binding sites and the site for cytochrome c. When
the reduced chinon binds to the side at the outside face, one
of the electrons is transferred to cytochrome c, which can
accept only one electron, and to the second binding site.
The two protons are released to the outside. The oxidized
chinon is now able to get back to the NADH oxidase or
alternatively to the other binding site of the cytochrome
becomes reduced again by the transfer of electrons from
the cytochrome c reductase and protons from the inside.
As is described below, reimport of 10 protons drives in
most bacteria synthesis of 3 ATP from ADP and phos-
phate. ATP has a physiological free energy of hydrolysis
of 50 kJ mol1 . To synthesize 3 ATP, +150 kJ mol1 are
thus needed. At a pmf of 200 mV, reimport of a proton
yields about 20 kJ mol1 (19.3 kJ/mol more precisely),
so 10 protons generate 200 kJ/mol, which is sucient to
synthesize 3 ATP and produce some entropy. Transfer of
an electron pair from NADH (Eo = 320 mV) to molecular
oxygen (Eo = +816 mV) yields E = 1236 mV or G = 2 F
1.236 V = 238.5 kJ mol1 . Pumping out 10 protons against
a pmf of 200 mV costs +193 kJ mol1 , leaving 45.5 kJ mol1
for entropy. The problem is that this calculation was done
with Eo values for molar concentrations of the substrates;
however, molecular oxygen is not soluble in water at 1 M
concentrations and reaches only concentrations below
1 mM, and in some environments only micrometer or
nanometer concentrations. Moreover, this value may
uctuate with time because of the physiological activity
and oxygen-consumption of the cell and changes in oxygen
availability. A 10-fold change in the oxygen concentration
calculated for an electron pair transferred results in a
decrease of the half cell potential for this acceptor of 30 mV.
So the real half cell potential of oxygen is below +726 mV
(1 mM) and may be only +636 mV (1 M) or +546 mV
(1 nM), decreasing the yielded energy in these three cases
down to below 202 kJ mol1 (1 mM), or 184 kJ mol1
(1 M) or 167.1 kJ mol1 (1 nM). At such oxygen concen-
trations, there is not sucient energy gained by the redox
reaction to export 10 protons. If the redox reaction would
be strictly coupled to proton export by proton pumps,
reverse electron transport would automatically occur are
low oxygen concentrations. Therefore, a dynamic coupling
between both processes is needed.
This dynamic coupling mechanism is provided by the Q
cycle. In Figure S1.6, 5 protons are exported by the pumping
mechanism of the NADH oxidase and the cytochrome c
oxidase, the remaining ve by the Q cycle. The rst two
protons are released when the ubichinon is oxidized at the
outer face of the cytochrome c reductase. If the oxygen
concentration is high, the ratio of oxidized to reduced
cytochrome c is also high and results in the actual E
dierence between chinon and cytochrome c according
to the Nernst equation. This allows the chinon to cycle
between both sites up to three times, transporting three
additional protons to the outside. With decreasing oxy-
gen concentration, the actual E between chinon and
cytochrome c decreases until no cycling at all happens and
only the rst two protons are released.

S1.2.10
The F1 F0 -ATPase
The F1 F0 ATPase is a primary, ATP-driven transport sys-
tem that is either able to form an ion motive force, in most
organisms a pmf, by hydrolyzing the energy acid anhydride
bond between the / phosphates of ATP, or to do the oppo-
site, fusing ADP and phosphate into ATP. Most organisms
use the enzyme for pmf-driven ATP synthesis but ferment-
ing bacteria usually create a pmf by the opposite process to
allow pmf-mediated transport processes.
The F1 F0 protein complex is composed of many subunits.
The membrane-integral F0 complex contains one a-subunit,
two b-subunits, and between 9 and 14 c-subunits. The last
value depends on the organism and the number is 10 in E.
coli. The c-subunits form a ring that can rotate inside the
membrane. The membrane-attached F1 complex contains
three , three , and one , , and subunit. The a-subunit
of F0 is the proton channel leading from the outside to the
cytoplasm but the channel is interrupted in the middle of
the protein so that protons from the outside, which are
driven by the pmf to the inside, cannot ow freely into the
cytoplasm. The two proton channels lead from the outside
to an access point in the middle of the membrane and from
a second access point in the middle of the membrane to the
inside. Both access points are adjacent to each other but
the proton cannot hop from one to the other by tunneling,
or the pmf would, of course, collapse, which would kill
the cell.
To move from one access point to the next, the protons
have to bind to a c-subunit and the c-ring has to rotate. So,
the protons use the c-ring as a merry-go-around, a carousel.
It is at the moment unclear if the c-subunit that carries a
proton has to move just one c-position to deliver the proton
again to the other opening or if the protons are allowed the
full merry-go-around through the membrane; however, the
charged acceptor of the c-subunit may become neutral by
the protonation process, allowing the respective c-subunit
to pass into the hydrophobic core of the membrane. After a
nearly full rotation, the respective c-subunit reaches the a-
subunit again and is able to release the proton. To get back
into the membrane, the c-subunit has to be protonated by
another proton from the outside part of the proton chan-
nel. This assumption would argue for the full merry-go-
around and against a one-position-only. But independent
of the ner details of action, in E. coli with its 10 c subunits,
import of 10 protons causes the rotation of 360 of the c-
ring.
Thus, the F0 part of the enzyme is in fact a proton pump
again with a conformational change, a rotation, causing
proton transport or vice versa. As the c-ring contains a
-axis in its middle, full rotation of the c-ring causes full
rotation of this axis, which connects also to the three alpha
and beta subunits that are arranged alternatively in the F1
part. Rotation of the -axis causes strong conformational
changes in this 3 3 head portion. The b-subunits of the
S1.2 Energy and Transport S21
F0 part connects to the -subunit of F1 , and this holds the
3 3 head and prevents it from rotating too, so that the
rotation of the -axis leads to the desired conformational
changes only.
The three alpha-beta parts of the F1 head portion contain
a binding site for ATP each but every site is always in
a dierent conformation: (i) for ADP + phosphate; (ii) a
close site that brings both molecules close together; and
(iii) for ATP. All three sites rotate between these three
conformation so that rst ADP and phosphate are bound;
second, the site is closed so that the concentration of water
at this position is zero, which leads to the formation of ATP
in the reaction ADP + phosphate ATP + H2 O. After this,
the individual site rotates to the ATP conformation and
ATP can be released.
So, the proton import drives a rotation, which leads to a
conformational change and formation of ATP by squeezing
water out of ADP and phosphate in a water-free microenvi-
ronment. The resulting ATP is the central energy-rich com-
ponent of all living organisms.
Although ATP synthesis by F1 F0 ATPases or related com-
pounds is used in dierent compartments and location in
bacterial, archaeal, or eukaryotic cells, this basic mode of
energy conservation is very similar in all living organisms
(Figure S1.7).
S1.2.11
Energy Pools in the Cell
So, the F1 F0 -ATPase has a central position in the energy con-
servation process in cells, either by using an ion motive force
to synthesize ATP or hydrolyzing ATP to build up an ion
motive force such as the pmf. The exchange ratio for this
energy transformation is determined by the number of c-
subunits in the F0 part, and this value depends on the par-
ticular organism.
Ion motive forces represent a physicochemical short-
termed energy pool. The pmf or other ion motive forces
can be used to drive a variety of processes: (i) rotation of
the bacterial agellum in swimming bacteria; (ii) other
mechanoproteins such as those used for bacterial gliding;
(iii) secondary transport; (iv) active transport across a
second, outer membrane in Gram-negative bacteria; (v)
reverse electron ow; and (vi) synthesis of energy-rich
compounds other than ATP.
The energy of the acid anhydrite bond in ATP is freely
used to synthesize other energy-rich bonds such as the
formation of ADP from AMP or that of the other three
important nucleotide-5 -phophates (NTPs), UTP, CTP,
and GTP. The NTP pool is the chemical medium-termed
energy-pool of the cell. All four NTPs govern dierent
processes: (i) ATP is responsible for the synthesis of basic
components of the macromolecules such as the amino
acids, and serves as energy donor for all house-keeping
functions; (ii) GTP controls protein biosynthesis and some
components of the information procession in cells; (iii)
S22 S1 Basic Biochemical Roots
Prokaryot Mitochondrion
(Escherichia coli)
H+ H+
ADP+Pi ADP+Pi
ATP ATP
Cytosol Periplasm Matrix IMS
Figure S1.7 Topology of ATP synthesis in bacteria and plant cell organelles. The ATP synthesis reaction is a universal one but is used
in dierent cellular compartments in bacteria (between cytosol or cytoplasm and the periplasm) or eukaryotic cell organelles. (Graphics:
D.Dobritzsch, G. -J. Krauss.)
UTP governs carbohydrate conversions; and (iv) CTP
those of some fatty acids. All four, however, are needed for
transcription, formation of RNA, and to amplify and read
out the genetic information of DNA. Moreover, when a cell
needs to divide, 2 -desoxy-NTPs are synthesized to allow
DNA replication.
Note a very important insight at this point: to under-
stand the general process of energy conservation in cells,
knowledge about all the pathways from a photon to ATP
and the other NTPs are needed. At the level of the NTPs,
the energy ow is nally coupled with the ow of infor-
mation stored as genes in the DNA to mediate synthesis
of macromolecules. Do note that there is an inherent
basic coupling, a balance, between the dierent modes of
macromolecule synthesis processes. Transcription needs
all four NTPs in sucient concentrations. If the cell needs,
for instance, more UTP for carbohydrate conversions, the
UTP concentration decreases, transcription slows down,
and with it protein synthesis at the ribosomes, translation,
which consumes nearly half of the cellular energy budget.
So, the cell can keep up by slowing down, and energy
is channeled into carbohydrate synthesis at the expense
of translation until this process is nished. The same is
true with fatty acids and CTP and metabolites in general
and ATP.
If sucient NTPs are present to allow growth but other
factors are lacking, for instance, sucient nitrogen, cells
are able to store energy in long-termed energy pools.
These can be carbohydrates such as starch or glycogen,
or hydrophobic substances such as fats and polyhydroxy
acids. The hydrophobic substances contain more hydrogen
equivalents than the carbohydrates and are thus a storage
compound not only for carbon and energy but also for
redox equivalents. In addition, some proteins may serve
as storage compounds for nitrogen. Polyphosphate can
be use to store phosphate, and metals may be stored
together with polyphosphate in cellular compartments
called acidocalcisomes. Finally, sulfur may be stored as
elemental sulfur or as thiol group in glutathione.
Chloroplast /
cyanobacterium
H+
ADP+Pi
ATP
Stroma Lumen IMS
S1.2.12
Life Styles
S1.2.12.1 Phototrophs
Phototrophs use photons to synthesize ATP via excitons,
redox-active compounds, an ion-motive force, the F1 F0
ATPase, and they use the NTPs to synthesize macro-
molecules. The carbon required for this process can come
from carbon dioxide CO2 . If a cell synthesizes most of
its macromolecules (>50%) from CO2 , its metabolism is
autotroph. The opposite, synthesis of the cell mainly from
organic molecules, is called heterotroph. If either process is
not predominant in a cell and that cell assimilates organic
molecules but xes CO2 nevertheless, this exception is
called mixotroph.
Some photrophs are photoheterotrophs or pho-
tomixotrophs but the most important ones on the
global scale are photoautotrophs. To assimilate CO2 into
biomass <CH2 O>, a redox donor DH2 is needed: CO2 + 2
DH2 <CH2 O> + 2D + H2 O. For the actual carbon xa-
tion reaction, NAD(P)H or reduced iron-sulfur cluster are
needed, which have a redox potential of Eo = 320 mV or
below.
Photoautotroph that use an inorganic substance as
electron donor for assimilation of CO2 are called pho-
tolithoautotrophs (Figure S1.8). The opposite from litho
(Greek for stone) would be organo, indicating the usage
of an organic electron donor, but it usually makes no
sense to assimilate CO2 with electrons coming from an
organic substance. In such a case, the respective organism
grows mixotrophically. Inorganic electron donors for
photolithoautotrophic organisms can be, for instance,
DH2 = H2 O, H2 S, NO2 , reduced Fe(II) or H2 , leading
to D = O2 , elemental sulfur, NO3 , Fe(III), or nothing,
respectively.
In case of H2 D = H2 O, the organisms are oxygenic
photolithoautotrophs. These are the cyanobacteria (Bac-
teria, Cyanobacteria), a subgroup of cyanobacteria called
prochlorales, and all chloroplasts of algae or plants, which

Photons
CO2 <CH2O>
2 H2D 2 D + H2O
Figure S1.8 The photolithoautotrophic life style. Light energy is har-
vested via an exiton, a redox compound with negative redox potential,
an ion motive force, and nally as ATP. The energy is used to reduce
carbon dioxide to biomass <CH2 O>, which requires a reduced electron
donor H2 D and produces water and 2 D.
ultimately are descendants of cyanobacteria. All these
dierent kinds of cyanobacteria have acquired during
evolution two dierent photosynthetic reaction centers, a
chinon-typed named PSII and an iron-sulfur-typed named
PSI. In PSII, light is harvested as excitons by phycobilins
in case of the real cyanobacteria (but not the prochlorales
or chloroplasts). In addition, in all oxygenic organisms, the
center contains a water-splitting manganese-containing
complex that oxidizes water into molecular oxygen and
four electrons after absorption of photons. So, water is the
electron donor for the special pair. Acceptor is a chinon
named plastochinon. The reduced plastochinon delivers
the electrons to a cytochrome-containing membrane-
bound protein complex, performing a Q-cycle; however,
the electrons are not transferred to a cytochrome c but
to a copper-containing plastocyanin, which is the elec-
tron donor for PSI. The second light reaction pushes the
electrons to a negative redox potential so that reduced
ironsulfur compounds and nally reduced NADPH/H+
can be produced. Moreover, during this noncyclical or
Z-scheme electron transport, a pmf is generated that
can be used to synthesize ATP via F1 F0 -ATPase. Electron
transport may also occur as cyclic electron transport that
involves only PSI and generates pmf but no NADPH.
Other, nonoxygenic or anaerobic photolithoautotrophic
organisms are bacteria belonging to the Chlorobiaceae
(Bacteria, Chlorobi) or to the Chromatiaceae (Bacteria,
Gammaproteobacteria). These bacteria contain either a
PSI-like iron-sulfur-type center or a PSII-like chinon-
type center without the water-splitting complex, and
electron transport is always cyclic. To produce NADPH,
they need the inorganic electron donor D, for instance,
H2 S, and reverse electron ow to decrease the redox
potential of these electrons to the needed NADPH level
of Eo = 320 mV. Photoheterotrophic/photomixotrophic
Chloroexaceae (Bacteria, Chloroexi) or Rhodospir-
illaceae (Bacteria, Proteobacteria) use cyclic electron
transport to conserve energy, assimilate organic substances
to some extent, and mix this reaction with the additional
xation of CO2 .
S1.2 Energy and Transport S23
An exceptional mode to conserve energy from photons
was realized in halobacteria (Euryarchaeota), a group
of the archaea. These salt-tolerant organisms produce
a pmf with light but without intermediate redox-active
compounds. Their photosystem is the membrane-bound
bacteriorhodopsin, which absorbs a photon with a retinal.
The exciton leads to a change of the conformation of the
retinal (trans to cis), coupled with a conformation change
of the protein, leading to proton export.
S1.2.12.2 Chemolithoautotrophs
Autotrophic life can also be powered by a chemolithotrophic
mode of energy conservation (Figure S1.9). This form of
life may be even more primordial than photoautotrophic
life because photons are dicult to harvest. Chemolithoau-
totrophs transfer electrons from a reduced electron donor
DH2 via an electron transport chain to an oxidized electron
acceptor A, conserve the resulting energy as pmf and
further on as ATP, and use this energy to reduce CO2 to
<CH2 O> with additional DH2 moieties.
Possible electron donor could be H2 , CO, reduced sulfur
compounds such as H2 S, reduced metals such as Fe(II)
or Mn(II), NH4 + , NO2 , or CH4 . All these compounds
originate in anaerobic environments by fermentation and
anaerobic respiration processes. Possible electron acceptors
may be O2 , NO3 , SO4 2 , CO2 , oxidized metals such as
Fe(III) or Mn(IV), NO3 , NO2 , or even organic substances
such as succinate, dimethylsulfoxide, trimethylamine-N-
oxid, or poly-halogenated compounds. Thermodynamics
decide which combination of donor and acceptor can be
used: the actual redox potential at the position of the cell as
calculated by the Nernst equation Eo = Eo + RT/(nF)ln
{[A]2 /([AH2 ] [D])} with R being the general gas constant,
T the temperature in (K), n the number of electrons
transferred, F the Faraday constant, and [A], [AH2 ], [D],
the concentrations of the acceptor, the reduced acceptor,
the donor, and the reduced donor, respectively. The Nernst
equation means that the actual dierence in redox potential
depends also on the concentration of the reactants. High
concentrations of the educts A, DH2 increase the redox
A AH2
2 H2D D
Energy
CO2 <CH2O>
2 H2D 2 D + H2O
Figure S1.9 The chemolithoautotrophic life style. To harvest energy,
electrons are transferred from a reduced electron donor H2 D (e.g., H2 ,
CO, CH4 , H2 S, NH4 + , Fe2+ ) to an oxidized acceptor A (e.g., O2 , NO3 ,
SO4 2 , CO2 ) and the energy is conserved as ATP via an ion motive
force. The energy is used to reduce carbon dioxide to biomass <CH2 O>,
which requires the reduced electron donor H2 D again and produces
water and 2 D.
S24 S1 Basic Biochemical Roots
potential, high concentrations of the products AH2 , D
decrease it. At n = 2 and 303 K, RT/(nF) = 13 mV. So,
a 10-fold increase in the concentration of a substrate
increases the actual Eo 30 mV, a 10-fold increase in the
concentration of a product decreases it to the same extent.
The actual E should allow proton export against a pmf of
200 mV, so E should be larger than 300 mV.
Usually, all electron donors can be used with molecular
oxygen as electron acceptor, and all electron acceptors with
molecular hydrogen as electron donor. The electrons from
the electron donor DH2 are usually transferred to a chinon
or to cytochrome c, depending on the individual redox
potential. The following respiratory chain is not so much dif-
ferent from that of organisms that respire with organic elec-
tron donors and may involve a Q-cycle using a cytochrome-
rich protein complex similar to complex III of the
mitochondria, and/or a cytochrome c oxidase. Alternatively,
the chinon compound may also donate its electron directly
to molecular oxygen or another electron acceptor. Other,
more complicated pathways, however, are also possible, for
instance, in methanogenesis by archaea, which is in fact a
H2 -dependent respiration with CO2 as electron acceptor.
To obtain suciently reduced compounds such as
NADPH for assimilation of CO2 , usually reverse electron
transport using DH2 as electron donor is required. From
the point of thermodynamics, only H2 may be able to
reduce NAD(P)+ directly; however the actual concentra-
tions of molecular hydrogen in the environment are very
low, predicting half-cell potentials more positive than
the Eo = 420 mV calculated for 1 M H2 . For this reason
molecular hydrogen in most organisms is assimilated by a
membrane-bound hydrogenase that delivers the electrons
to the chinon pool. Only a very few bacteria possess a
soluble hydrogenase that is able to reduce NAD+ directly,
thus avoiding the necessity of reverse electron ow.
Chemolithotrophic organisms reside in environments
with gradients between oxidized and reduced compounds
such as the oxic-anoxic transition zone (OATZ) in aquatic
ecosystems as well as in soil. Here, they use the reduced
compounds from anoxic zones as electron donors. Their
respective reduced electron acceptor may diuse to the
next transition zone and the electrons may be transferred
to yet another electron acceptor until the electrons reach
molecular oxygen to form water. Thus, chemolithotrophs
are part of a chain of organisms that transfer electrons
in multiple steps from organic substances in anoxic envi-
ronment ultimately to molecular oxygen. In addition,
important chemolithoautotrophs in oxic environements are
the ammonium- and nitrite-oxidizing nitriers.
S1.2.12.3 Chemoorganoheterotrophs
There are only a few combinations between photo-
/chemolitho-/organo-auto-/heterotrophs that are
sensible and are realized by some living organisms. In
chemoorganoheterotrophs, organic substance <CH2 O> is
degraded, usually to CO2 in the reaction H2 O + <CH2 O>
CO2 <CH2O>
A AH2
Energy
<CH2O>-Assimilation
Figure S1.10 The respiratory chemoorganoheterotrophic life style.
To harvest energy, biomass <CH2 O> is degraded to carbon dioxide and
the resulting electrons are transferred to external electron acceptors A
(e.g., O2 , NO3 , SO4 2 , CO2 ) by an electron transfer chain. The energy is
used to assimilate other parts of the biomass <CH2 O>.
COOH <CH2O>
<CH2O> CH3
Energy
<CH2O>-Assimilation
Figure S1.11 The chemoorganoheterotrophic life style by fermen-
tation. Biomass <CH2 O> is disproportionated, leading to oxidized (car-
bon dioxide, COOH) and reduced (CH3) carbon atoms in the nal fer-
mentation produces (e.g., lactate, ethanol/CO2 , butyrate, propionate).
This reaction needs no external electron acceptor but conserves less
energy compared to a respiration. The energy is used to assimilate other
parts of the biomass <CH2 O>.
CO2 + 2 2 [H]. The electron equivalents released by this
reaction can be transferred in by a respiratory chain to
an external electron acceptor, a process called respiration
(Figure S1.10). Alternatively, an internal electron acceptor
may be generated by the disproportionation of the original
substrate in a process called fermentation (Figure S1.11). In
addition to this dierence, respiration allows conservation
of more energy than fermentation and uses mainly an
electron transport chain for this process.
Possible electron acceptors are the same as those for
chemolithoautotrophic organisms: O2 , NO3 , SO4 2 , CO2 ,
oxidized metals such as Fe(III) or Mn(IV), NO3 , NO2 ,
or even organic substances such as succinate, dimethyl-
sulfoxide, trimethylamine-N-oxid, or poly-halogenated
compounds. Depending on the individual acceptor,
reduced chinons or in a few cases reduced cytochrome
c function as electron donors for a terminal oxidase. The
resulting energy is conserved as pmf or another ion motive
force, and ATP is synthesized by F1 F0 -ATPase. While
in chemolithoautrophic organisms the reduced electron
donor has a dual function, providing the electrons either for
energy conservation or for xation of CO2 , the organic sub-
strate plays a similar role in chemoorganoheterotrophs: this
substance can either be degraded to CO2 and NADH, and
the latter used to provide electrons for energy conservation

by respiration, or the substance can be used as an educt
to synthesize a metabolite such as an amino acid. In an
aerobic respiration at sucient oxygen concentrations,
about half of a substrate such as glucose is used for energy
conservation while the other half is assimilated.
In fermentation, the energy yield per mol of substrate is
much lower compared to respiration because the substrates
are not completely degraded to CO2 because of the neces-
sity of an internal electron acceptor. Therefore, fermenting
organisms release fermentation products that still contain a
lot of energy. To compensate for this, they metabolize more
substrate per time unit than respiring organisms and pro-
duce more heat.
In syntrophy, by interspecies hydrogen transfer anaer-
obically respiring organisms use the molecular hydrogen
as substrate that is produced by adjacent fermenting
S1.2 Energy and Transport S25
organisms. By doing so, the respiring organisms decrease
the concentration of molecular hydrogen, allowing fer-
menting organisms to release superuous redox power as
molecular hydrogen and subsequently to conserve more
energy per mol of substrate. Thus, syntrophy is an ecological
symbiosis, which allows an ecient degradation of organic
substances in the absence of molecular oxygen.
Because of the low redox potential of their cellular
components, syntrophic organisms are very sensitive to
molecular oxygen. The reduced acceptors of anaerobic
respiration processes diuse away from the anaerobic
microenvironments of syntrophic partners and may serve
as electron donors for chemolithoautotrophic organisms.
These consume the molecular oxygen coming from the
other side and thus protect these anaerobic environments
eciently against oxygen.
S26 S1 Basic Biochemical Roots
S1.3
Basic Biochemistry
Overview
This section is a concise overview of the basic biochemical
pathways in living organisms including fermenting
bacteria.
S1.3.1
Organization of the Overall Metabolism
The second section in this chapter described the energy
ow in living organisms. Phototrophic organisms
use photons, chemolithoautotrophs the redox dier-
ences between inorganic compounds, and respiring
chemoorganoheterotrophic the redox dierence between
an organic and an inorganic compound to generate an
ion motive force, in most cases a pmf, to drive ATP-
synthesis. Fermenting chemoorganoheterotrophs couple
ATP-synthesis directly to biochemical reactions. Other
NTPs (GTP, CTP, UTP) are produced with ATP, and all
NTPs are used to transcribe the genetic information of the
DNA into RNA, thus coupling the energy ow of the cell to
the information ow.
The resulting RNA is either active as it is, for example,
as rRNAs, the central components of the ribosomes, or
as tRNAs that transport amino acids to the ribosome
for translation. The mRNAs provide the information how
amino acids are to be assembled into a polypeptide chain
during translation. That way, synthesis of two of the four
classes of cellular macromolecules, nucleic acids and
proteins, are part of an information ow that is a direct
consequence of the energy ow of the cell. Moreover,
generation of both classes of macromolecules is interwoven
with each other. Proteins are needed for DNA replication,
repair, stabilization and packaging, transcription, tran-
scription control, and RNA degradation, and they form the
ribosomes together with the rRNAs. On the other hand,
RNAs control the information ow from DNA to proteins,
including the formation of the peptide bond and even some
steps in protein targeting. Nevertheless, a very simple way
to describe a living cell is a bag of ribosomes that makes a
new bag of ribosomes.
Proteins alone are responsible for transport, sensing of
external signals, many regulatory processes, formation of
metabolites including building blocks, and synthesis of the
other two classes of macromolecules, carbohydrates, and
hydrophobic substances such as phospholipids. Proteins
can fold into structures that contain essentially water-free
reaction centers much easier than RNA molecules, and

are thus better catalysts. Catalytically active proteins are
designated enzymes.
The enzymatic reactions of a living cell form a compli-
cated network of individual reactions, mostly located in the
cytoplasm of bacteria or archaea, but maybe distributed
to dierent organells in an eukaryotic cell. Despite this
complexity, biochemistry is simple. A few mechanisms are
responsible for most reaction: (i) carbonyl-based reactions;
(ii) hydride transfer; (iii) nucleophilic substitutions; (iv)
additions to double bonds; and (v) elimination to form
double bonds. The more complicated reactions are stan-
dardized by cofactors. A central backbone of biochemistry,
the fructose-1,6bP-pathway, is used for conservation of
energy by degradation of organic substances (catabolism)
and for biosynthesis of new building blocks (anabolism).
Additional circles or shunts are attached to this central
backbone and serve special needs, most important the
tricarbonic acid cycle (TCA cycle), which may also work
as a two-forked double shunt.
This section starts with enzymes and cofactors, investi-
gates the F1,6bP-pathway and how easy the reactions in this
pathway can be understood, and leads on to the attached
metabolic cycles and shunts. It concludes with an overview
of genomes and evolution.
S1.3.2
Enzymes and Coenzymes
Enzymes are proteins that catalyze a biochemical reaction.
For any kind of a chemical reaction, one or more substrates
have to form a transition state between educt(s) or sub-
strates and product(s). This transition state is a state of high
energy. As the energy states of the molecules in a system has
a statistical distribution, the MaxwellBoltzmann distribu-
tion can be used to calculate the probability for a molecule
in a population to reach the energy of the transition state.
The higher this energy is, the lower the probability that
a molecule reaches it, and the slower, consequently, the
reaction rate. Here, catalysts come into play. They react
with the molecule(s), circumventing the transition state of
high energy by using one or several transition states with
considerable lower energy, thereby increasing the rate of
the chemical reaction. A catalyst is usually regenerated after
its reaction cycle has been completed.
Enzymes are catalysts that bind their substrate(s) in a
three-dimensional substrate-binding center. By providing
cofactors, prosthetic groups or active amino acid residues
of the protein, they circumvent high-energy transition
states and substitute them with those of lower energy. An
example would be the F1 F0 -ATPase (see Section S1.2). The
three active sites of this protein complex rotate between
three conformations. In one, ADP and phosphate are
bound, in the second, the substrate-binding center is closed
and ADP/phosphate are brought very close to each other in
a water-free environment so that ATP can be formed, and
in the third, ATP is released. Similar to transport proteins,
S1.3 Basic Biochemistry S27
enzymes show substrate-saturation, ideally following the
MichaelisMenthen equation (see Section S1.2).
The only dierence between cofactors and prosthetic
groups of a protein is that cofactors may leave the enzyme
after the reaction while prosthetic groups stay. So, cofactors
have to be regenerated by a second enzyme but prosthetic
groups by a subsequent second reaction of the same
enzyme. Nevertheless, cofactors (Figure S1.12) and pros-
thetic groups similarly are both standardized biochemical
reaction partners.
ATP and the other NTPs have a D-ribose in the ring
form at their center. The ring is formed by an attack of the
C4 hydroxide on the aldehyde, carbonyl-C1 , and xed
by an exchange of the C1 OH against an N-glycosidic
CN bond between the C1 atom of the ribose and a
nitrogen of the attached cyclic purine (adenine, guanine) or
pyrimidine (uracil, cytosine) base, forming a nucleoside. In
a nucleoside, the atoms of the ribose are numbered C1 to
C5 with the indicating the dierence between a ribose
atom and a purine of pyrimidine atom. The C5 atom of the
ribose carries the rst -phosphate moiety in a low-energy
ester bond, resulting in a nucleotide mono-phosphate. The
second and the third -phosphates are both bound as
high-energy acid anhydrites, making NTPs and especially
ATP to very important energy-rich compounds of the
metabolism with a free hydrolysis energy in vivo of about
50 kJ mol1 . Coupling any other reaction to hydrolysis of
one or both of these acid anhydrite bonds releases a lot of
energy as heat and drives the respective reaction into the
desired direction.
NAD+ (nicotinic acid amide dinucleotide) is composed
of two AMP moieties fused by an acid anhydrite bond of the
two respective -phosphates; however, instead of a second
adenine base, one ribose carries a nicotinic acid amide. As
an aromatic ring, the nitrogen atom in the N-glycosidic
bond is positively charged, The carboxy-amide group in
meta-position to this nitrogen further delocalizes the ring
electrons along all three atoms (CON) of this group
and stabilizes a partial positive charge in ortho-position
to itself and in para-position to the ring nitrogen. This
positively charged ring carbon can easily accept a hydride
H , a proton plus an electron pair, and release it again.
Such a reaction involves formation or usage of a proton H+
going to or coming from the solvent water and has a redox
potential of Eo = 320 mV. NADP+ has the same redox
potential but carries an additional phosphate ester at the
C2 atom of the adenosine-carrying ribose. While NAD+
is involved in catabolism and hides for the respiratory
chain in respiring organisms, NADP+ is usually involved in
anabolism; however, several catabolic reactions produce
NADP+ for anabolic purposes. Transhydrogenases trans-
form an equilibrium between NADH and NADPH, thus
connecting both hydride pools.
FAD looks like NAD+ but the second D-ribose is in the
open conformation and carries a avin instead of a nicotinic
acid amide group. FAD and the related FMN can accept (or
S28 S1 Basic Biochemical Roots

O NH2 N
NH
H NH2
N
H N N N O N
NH2
N N OH
N N
NADH O FAD
OH N
O
O OH
O HO N
HO O O O O
P P O
OH O
O O
OH O P
P O

OH
O O HO
O
NH2
N
N

N SH
N
OH
ATP O
HN O
OH

O O O
O O O O
P P P
O O O
HN
OH
O

O
Coenzym A O

P O
O

O P O O
H P O O O O

P O

N
+ H
S O O O P O
Thiamine diphosphate O
O
+
N N
HO O
NH2
N N

N
N
NH2

O H H
H H O S COOH

O P
O H H
O
Pyridoxal phosphate O Biotin HN NH
+
N
H O

Figure S1.12 Structure of selected cofactors.

release) hydride moieties H to one of the nitrogen atoms of the 3 -phospho-ADP. A derivative of HS-CoA is the
in the avin triple ring, and a proton to another ring nitro- 4 -phospho-pantethein that is attached to a serine residue
gen. Alternatively, they can be reduced by an electron pair of a small very acidic protein, the acyl-carrier protein ACP.
and take up two protons to form the reduced avin ring. It should be noted that all these ADP-like cofactors are
That way, FAD or FMN are intermediates between hydride- able to bind to a similar fold in the enzyme, the nucleotide-
transfer cofactors such as NADH and acceptors of electrons binding or Rossmann-fold (Figure S1.13). Of course, the
only such as ironsulfur centers. individual folds for these dierent cofactors are dierent
Another but more complicated derivative of ADP is but by using modications of the same motif, evolution of
coenzyme A, HS-CoA. The ADP moiety carries an addi- enzymes that bind these cofactors should have been an easy
tional phosphate ester at the C3 atom of the ribose. The event.
important functional group of HS-CoA is the thiol group Thiamindiphosphate or thiaminpyrophosphate (TPP) is
that can attach all kinds of organic acids in an energy-rich essentially a CH carbon in a double bond with a positively
thioester bond to activate these acids and to carry them charged quaternary nitrogen (-amin) and in a single bond
from one enzyme to the next one. This thiol group is with an electron-rich sulfur (thi-) on the other side. The
part of 2-mercaptoethylamine (1-amino-2-thio-ethan) CH bond of this carbon is very acidic so that TPP serves
that is in an amide bond with pantothenic acid leading to as standardized CH acidic bond.
pantethein. Pantothenic acid is 2-hydroxy-3,3 -dimethyl- Liponic acid is a ring structure containing two adjacent
4-hydroxy butyric acid in an amide bond with 3-amino sulfur atoms. A spacer attaches the ring in an amide bond
propionic acid. Pantethein serves as a spacer for the thiol to the terminal amino group of a lysine in enzymes. The
group and is attached in an ester bond to the -phosphate two sulfur atoms are in a single bond with each other in the

Figure S1.13 The Rossmann fold.
oxidized form but contain a hydrogen atom each instead in
the reduced form. Liponic acid can oxidize compounds that
cannot be oxidized by the removal of a hydride ion.
Biotin attaches itself also by a spacer and an amide for-
mation to amino groups of amino acyl residues of proteins,
for instance, to lysine side groups. The biotin double ring
contains a sulfur in one ring and two nitrogen atoms in the
other. One of these can accept a carbon dioxide so that biotin
serves as carbon dioxide transferring prosthetic group.
Cobalamine is a complicated compound carrying a
cobalt-containing heme group at its center. This factor is
used to rearrange CC and CH bonds.
There are other cofactors. Pyridoxal phosphate binds
amino acids as Schi base or imine. Tetrahydrofolate can
bind C1 -compounds and transfer them to oxidation and
reduction reactions. Electron carriers of the respiratory
chain such as cytochromes, iron-sulfur centers and cop-
per centers as well as chlorophyll and carotinoids have
already been mentioned in Section S1.2. Methanogenic
archaea possess a variety of interesting cofactors involved
in methanogenesis such a F420, a hydride-transferring
deaza-avin, or F430, a nickel-containing heme. Neverthe-
less, the most important cofactors necessary to understand
the central metabolic pathways are those listed above.
S1.3.3
The Backbone: Fructose-1,6-bP Pathway
S1.3.3.1 Overview
The fructose-1,6-bis-phospate or F1,6bP pathway
(Figure S1.14) is the central backbone of the cellular
metabolism. It connects glucose with pyruvate and can be
used in either direction as glycolysis (catabolic direction)
or gluconeogenesis (anabolic direction). Anabolic reactions
for many buildings blocks such as amino acids, glycerol for
phospholipids, and monosaccharides for cell wall and stor-
age compounds originate here and most sugar degradation
S1.3 Basic Biochemistry S29
pathways lead into the F1,6bP pathway. Moreover, other
metabolic cycles or shunts are attached to it (Figure S1.15).
The endpoints of the F1,6bP pathway are glucose-
6-phosphate (G-6-P) on the one hand and pyruvate
on the other. Milestones are the F1,6bP aldolase reac-
tion to glycerol-aldehyde-3-phosphate (3GAP) and
dihydroxyacetone-phosphate (DHAP) that connects hex-
oses with trioses, and the 3GAP dehydrogenase reaction
that connects trioses with organic acids. To understand
this pathway, we start with glucose and move from here
downward to pyruvate. We close with a glance at the fate of
pyruvate. Note that most reactions in the F1,6bP-pathway
are carbonyl reactions.
S1.3.3.2 Glucose
Glucose is a hexose, a carbohydrate with six C atoms. Other
carbohydrates are trioses (C3), tetroses (C4), pentoses (C5),
and heptoses (C7). Moreover, glucose is an aldose because
the carbonyl function is an aldehyde at the C1 atom. Glu-
cose is more precisely D-glucose. It contains four optically
active carbon atoms from C2 to C5 and the conguration
of the C5 atom is in D, that of the others in D, L, D from
C2 to C4, respectively. In aqueous solution, only a few
percent of the glucose molecules are in an open, linear
conformation. In the majority of the molecules, the oxygen
of the C5 carbon has attacked the C1 carbonyl group,
forming a ring structure, glucopyranose. The reaction,
hemiketal-formation, is a special form of the hemiacetal
formation resulting from an attack of an alcohol group on
an aldehyde (RHCO + ROH RHCOHOR). In the
glucopyranose ring, the C6 group and the alcohol functions
of the C2 to C4 atoms are equatorial, standing away from
the pyranose ring. Ring closure produces two possibilities
for the resulting hydroxide function of the C1 atom. In
the conformation, the OH group is also equatorial while
in , it is axial and close to the carbon atoms of the ring.
Therefore, most (two-third) of the glucose in an equilibrium
(mutorotation equilibrium) is in the -D-conformation.
It should be noted that D-glucose is thus the hexose
with the lowest energy in the ring conformation and its
mirror form L-glucose, consequently, that with the highest
energy. Because of this, most carbohydrates and their
derivatives in the metabolism are in the D-conformation,
and most amino acids, resulting from a nucleophilic SN2
substitution (and Walden inversion) of a OH group at
the C2 atom of a carboxylic acid by an amino group, are
in the L-conformation. There are seven D-hexoses that
are stereo-isomers of D-glucose. The most important of
these, D-mannose (LLDD conformation) and D-galactose
(DLLD), have only one alcohol group axial. The other ve
stereo-isomers (D-allose, D-altrose, D-gulose, D-idose, and
D-talose) are rare.
Glucose enters metabolism from the outside by the
glucose uptake systems or after the hydrolysis of a glucose-
containing storage compound such as starch or glycogen.
It is immediately phosphorylated to G6P by hexokinase
S30 S1 Basic Biochemical Roots

Energy investment phase Energy generation phase

O

OH O
HO
Glucose Glycerinaldehyde-3-phosphate OH
OH
OH CH2O-P

CH2OH GAPDH
Pi
ATP HK ADP
O 2 NAD+ 2 NADH/H+
OH O
P-O
HO
Glucose-6-phosphate OH 1,3-Bisphosphoglycerate OH
OH CH2O-P
CH2O-P
PGI 2 ADP PGK 2 ATP
CH2OH
O
COOH
HO
Fructose-6-phosphate 3-Phosphoglycerate OH
OH
OH CH2O-P
CH2O-P
ATP PFK ADP PGM
CH2O-P
O
COOH
HO
Fructose-1,6-bisphosphate 2-Phosphoglycerate O-P
OH
OH CH2OH

CH2O-P
ALDO ENO

COOH
CH2O-P
Dihydroxyacetonephosphate Phosphoenolpyruvate O-P
O
CH2
CH2OH

TIM 2 ADP PK 2 ATP

O
COOH
OH O
Glycerinaldehyde-3-phosphate CH2O-P Pyruvate CH3

Figure S1.14 Fructose-1,6-bP pathway (glycolysis). 3-phosphate dehydrogenase; PGK Phosphoglycerate kinase;
(HK Hexokinase; PGI Glucose 6-phosphate isomerase; PGM Phosphoglycerate mutase; ENO Enolase; PK Pyruvate
PFK Phosphofructokinase-1; ALDO Aldolase; kinase) (Graphics: D. Dobritzsch.)
TIM Triosephosphate isomerase; GAPDH Glyceraldehyde

or during the uptake process by a phosphotransferase
system (PTS) group translocation system (see below at
Phosphoenolpyrvate, PEP). Note the G6P is still able to
perform mutorotation and thus allowed to form both kinds
of the ring structure and the open linear conformation. In
contrast, glucose-1-phosphate G1P attaches the phosphate
ester to the OH-group of C1, which xates the respective
conformation, resulting in -D-G1P or -D-G1P. Both
compounds have a dierent fate. If D-G1P polymerizes
in a 14 glycosidic bond, again a hemiacetal, the macro-
molecule is kinked between the glucose moieties and forms
a helix. The molecule is starch or glycogen, depending on
the organisms (plant versus animals) and the amount of
branches, (14) polymers attached to C atoms other than
C1 or C4. In contrast, the polymers of D-G1P are straight
linear macromolecules that tend to para-crystallize: cel-
lulose, an important cell wall material in plants and other
organisms. If the C2 atoms of cellulose carry an N-acetyl
group, the result is chitin, cell wall material in insects and
some fungi. Finally, if every second 2-N-acetyl-glucosamin
contains a lactyl-ether (muraminic acid) and a more or
less cross-linked short polypeptide chain, this leads to
murein or peptidoglycan, the most important cell wall
material in bacteria. Thus, the fate of D-G1P is that of a
building block for cell walls, and of D-G1P that of a storage
compound.
Both polymers, starch and cellulose, are synthesized by a
nucleophilic substitution: a free electron pair of the O atom
of the C4OH group attacks the nucleus of C1, releasing the
phosphate.
A note to remember at the end of this section: phospho-
rylation of glucose at the beginning of the F1,6bP-pathway
has very important reasons. First, nearly all metabolites are
charged. This allows them to bind rmly to the substrate-
binding sites of enzymes by ion-ion-interactions and a
correct positioning there. Second, a charged substrate
does not diuse across a biological membrane to get lost
but can instead be attracted to the correct position at the
surface of enzymes that lead as a kind of mouth to the
substrate binding site. If the substrate is negatively charged,
 S1.3 Basic Biochemistry S31

Raffinose, stachyose, Fructans

verbascose (carbohydrate
(osmolyte) storage)

Galactinol
Sucrose
(carbohydrate
UDP
storage)
Starch
myo-inositol (carbohydrate Pi
storage)
Sucrose- Mannitol
6-phosphate (osmolyte)
UDP-galactose
UDP- Pi

Glucose- glucose
Fructose- Mannitol- Mannitol-
UDP-glucose 1-phosphate 6-phosphate 6-phosphate 1-phosphate

UTP Glucose
Glucose-
Glucose
6-phosphate

UDP-glucose Glycolysis Calvin cycle Pentose

phosphate
cycle
Trehalose-
6-phosphate
Gluconeogenesis

Pi
Trehalose
(osmolyte)

Figure S1.15 Primary sugar routes in plants. (Graphics: G.-J. Krauss, D. Dobritzsch.)

this mouth usually carries positive charges at the surface.
Third, in case of the F1,6bP pathway, the chemical reaction
equilibria of the aldolase and the triose phosphate isomerase
reaction would favor fructose-1,6-bisphosptate F1,6bP and
DHAP, respectively. Without at least one phosphorylation,
the concentration of glycerinaldehyde-3-phosphate 3GAP
would be lower than one molecule per cell in the ow
equilibria of the F1,6bP-pathway. Formation of a phosphate
ester in G6P from ATP releases about 30 kJ mol1 as heat,
driving the reaction equilibria to a point that sucient
3GAP is available for the following reactions. There is
even more 3GAP if a second ATP is sacriced and most
organisms do exactly that, but the fact is that some archaea
do not. These use F-6-P for the aldolase reaction and
phosphorylate the dihydroxyacetone later on. Finally, a
glucose-specic reason was given: the position of the
phosphate residue marks the fate of the glucose moiety as
with cell wall (D-G1P), storage (D-G1P), or degradation
(G6P) because the next step in the F1,6bP-pathway cannot
happen with G1P; it is possible only with G6P.
S1.3.3.3 Hexosephosphate-Isomerase
In a carbonyl-reaction, G6P is isomerized to fructose-6-P
(F6P). Fructose is a keto-hexose with the C-atoms C2 to
C5 in the keto, L, D, D conformation, respectively. The
aldose mannose can also isomerize to fructose. The other
three, rare, D-2keto-hexoses are D-psicose, D-sorbose,
and D-tagatose. As other carbonyl-groups, that of glu-
cose or fructose, can tautomerize into an enol form: the
carbonyl-oxygen that carries a negative partial charge
attacks a hydrogen atom of one of the adjacent CH acidic
carbon atoms forming an alcohol group (-ol). The now
free electron pair of the hydrogen-looser lls in and
forms a new double bond between the two carbon atoms
(CHHCO C=HCOH, not all bonds of the carbon atoms
shown). This reaction happens between the C1-aldehyde
and the C2 alcohol, leading to an intermediary enol form
that is common between glucose, mannose, and fructose.
The only job the enzyme has to do is to protonize the
carbonyl-O and accept the proton from CH-acidic car-
bon, or vice versa in the other direction. If the proton added
in this other direction to the future CH-acidic carbon
comes from the top or from the bottom decides if glucose of
mannose comes out o the reaction. The free energy of the
reaction G6P F6P is +1.68 kJ mol1 , so the equilibrium
between both compounds is about 2:1.
S1.3.3.4 Phosphofructokinase
A second phosphorylation step (F6P + ATP F1,6bP +
ADP, 14.3 kJ mol1 ) moves the equilibrium to F1,6bP and
further degradation products by a factor of about 292-fold.
Moreover, the products of the following aldolase reaction
are both phosphorylated and, therefore, charged. Finally,
the two negatively charged phosphate residues drag the
S32 S1 Basic Biochemical Roots
F1,6bP molecule by ionic repulsion into the open linear
conformation, which makes the aldolase reaction much
easier.
The kinase reaction is also a carbonyl reaction. The oxy-
gen of the C1 atom attacks the carbonyl-analogous (P=O)
phosphorous atom of the -phosphate of ATP, leading to
transfer from the ATP to the fructose and to the formation
of a low-energy ester from a high-energy acid anhydrite. Do
note again that G6P cannot perform this reaction because
it does not carry an alcohol group at C1 but an aldehyde, so
that the isomerase reaction has to happen before the (phos-
phofructo)kinase reaction.
Phosphofructokinase is a catabolic enzyme involved ex-
clusively in glycolysis. The back-reaction (F1,6bP + ADP
F6P + ATP) would have a bad ow equilibrium, 291:1 as
stated above. So, this reaction is unlikely to occur. Gluco-
neogenesis uses the anabolic hexose diphosphatase instead
that splits o the phosphate (16.8 kJ mol1 ). This is also a
carbonyl reaction; a free electron pair of the oxygen of water
attacks the P nucleus of the C1-phosphate group leading to
hydrolysis.
It would be a disadvantage if a cell allows both processes
to occur simultaneously. In a futile cycle, ATP would
be hydrolyzed in the presence of catalytical amounts of
F6P or F1,6bP. This must not happen, so activity of both
enzymes is strictly controlled. In other words, this is a
signicant checkpoint for the cellular biochemistry to
control if the F1,6bP pathway ows in the glycolysis or the
gluconeogenesis direction.
S1.3.3.5 Fructose-Diphosphate-Aldolase
This is the next carbonyl reaction, best understood when
examining the condensation reaction. In DHAP, the C2 is a
carbonyl; it carries a keto group. Thus, C1 and C3 are CH-
acidic but this does not matter for C3 because this atom car-
ries a phosphate ester (probably used to position the DHAP
in the aldolase enzyme). If the proton of the CH-acidic C1-
atom of DHAP is subtracted, the resulting free electron pair
is free to attack the C1-carbonyl C-atom of 3GAP, forming
a CC bond. The former carbonyl-oxygen receives a proton
and the reaction is completed. Some aldolases carry a diva-
lent metal cation in the reaction center that complexes the
carbonyl-O of the C1 group of 3GAP, facilitating the attack
from the CH acidic C1 of DHAP. Alternatively, in other
kinds of aldolases, DHAP is attached via a Schi base to
a lysine residue of the enzyme, which makes the C1-atom
CH acidic nevertheless. The Go is +24.1 kJ mol1 , lead- S1.3.3.8 Phosphoglycerate Kinase
ing to a ow equilibrium that is 14 280-fold on the side of
F1,6bP.
S1.3.3.6 Triosephosphate Isomerase
The products of the aldolase reaction, D-3GAP and DAHP,
are trioses, one of the two possible aldo-trioses (the other
would be L-3GAP) and the only possible keto-triose. The
triosephosphate isomerase reaction is thus a carbonyl
reaction similar to the hexosephosphate isomerase, again
using the enol form (double bond between C1 and C2) as
intermediate of both carbonyl compounds. The Go is
further +7.69 kJ mol1 in the direction of 3GAP, again 21-
fold in the wrong direction. Without the ATP-dependent
phosphorylation steps upstream, the resulting concen-
tration of 3GAP would thus be very low, far below one
molecule per cell (= 0.5 nM in a bacterial cell), even at an
initial concentration of 300 M glucose.
3GAP can be reduced to glycerol-3-phosphate, which is a
building block for (phospho-)lipids. This is a carbonyl reac-
tion with a hydride transferred from NADH to the carbon of
the C1 aldehyde and protonation of the previously double-
bound oxygen.
S1.3.3.7 Glyceraldehyde Phosphate Dehydrogenase
This is the central reaction of energy conservation for
lactic acid bacteria performing homolactate fermentation,
for Saccharomyces cerevisiae during anaerobic alcohol
fermentation and for muscles when they become anaerobic
during an extensive workout. It is a carbonyl-reaction again,
this time augmented by a hydride-transfer. In the hydride
transfer, which is actually an oxidation of the carbonyl-C1
to a carboxyl-C1, in an exergonic process (43.3 kJ mol1 ),
the released energy is conserved in an energy-rich mixed
acid anhydrite bond (+49.6 kJ mol1 ). The resulting Go
is +6.3 kJ mol1 (12-fold toward 3GAP) and the follow-
ing exergonic reaction is needed to drag the product
1,3-bisphoglycerate (1,3bPG) away during glycolysis.
The reaction itself is very smart. The hydrogen group
of the C1-aldehyde cannot be transferred as hydride
group to NAD+ because the carbon is partially positively
charged. So, the thiol goup of a cysteine residue attacks
the C1 carbonyl of 3GAP, leading to a thio-half-acetal:
E-SH + HC=O E-S-HCOH (one bond of the carbon not
shown). The C1 carbon is close to the electron-rich sulfur
because of the low dierences in electronegativity in a near
covalent bond. The hydrogen atom can consequently be
removed and transferred as hydride group to the previously
bound NAD+ leading to NADH. Release of a proton H+
from the OH-group restores the double bond and yields a
thioester E-S-C=O (one C-bond not shown). This is also
an energy-rich bond. A phosphoclastic split of this bond
leads to the resulting mixed acid anhydrite at C1: an oxygen
of phosphate attacks the C1 carbon as nucleophile and
detaches the 1,3bPG from the enzyme.
To yield ATP from the previously conserved energy-rich
bond, the phosphate from the C1 mixed acid anhydrite of
1,3bPG is transferred to ADP, forming 3-phosphoglycerate
3PG and releasing 18.9 kJ mol1 . This reaction also
removes 1,3bPG as product from the previous dehy-
drogenase reaction. Both reaction together have a Go
of 12.6 kJ mol1 , so a 149-fold concentration of 3-PG
compared to 3GAP. The kinase reaction is a carbonyl-
reaction and has been outlined above.

S1.3.3.9 Phosphoglycerate Mutase
At this point, the energy conservation of the glycolysis
has occurred but the two phosphate groups that were
used for activation have to be regenerated; however, the
two molecules of 3PG carry the phosphate residue in
a low-energy ester bond. In two interesting steps they
are moved up to a free hydrolysis energy of close to
80 kJ mol1 .
The rst of these steps is the phosphoglycerate mutase
that changes 3PG into 2PG (2-phospho-glycerate). In an
activation step, one ATP is needed to form a catalytically
amount of 2,3-bis-phospho-glyerate, 2,3bPG, which is
bound to the mutase. Now, the mutase binds 3PG, transfers
the phosphate group from the C3 of 2,3bPG to the C2 of
3PG, yielding 2PG and again 2,3bPG. The 2PG is released,
another 3PG bound, and the reaction performed again and
again. Of course, this reaction runs in either direction. In
the direction of 2PG, Go is +4.45 kJ mol1 , so the ratio of
3PG:2PG is 6:1. The reaction is again a carbonyl reaction
with an electron pair of the oxygen of the OH group attack-
ing the P nucleus of the phosphate group to be transferred.
S1.3.3.10 Phosphoglycerate Enolase
In the second step, water is eliminated. Go is +1.85 kJ mol1 ,
so the ratio of 2PG to the product PEP is 2:1. The reaction
is no carbonyl reaction for a change. Enolase depends on a
divalent metal cation such as Mg2+ or Mn2+ . The reaction
can be understood from the other direction. In PEP, a
conjugated double bonds exists between the carboxyl group
at the C1 position and the double bond between C2 and C3.
Protonation of the carboxyl-C1 leads to a partial positive
charge of the C1 carbon. In a mesomeric description, this
leads to a double bond between C1 and the CH-acidic
C2, and to a formal positive charge at C3. So, a hydroxide
group, previously bound to the metal cation, can be added
to C3 of PEP, and protonation of C2 leads to 2PG.
S1.3.3.11 Pyruvate Kinase and Phosphotransferase Systems
(PTS)
The phosphate group of PEP can now be transferred in a
kinase reaction to ADP leading to pyruvate, ATP, and the
release of 31.5 kJ mol1 . Together with the in vivo hydrol-
ysis energy of ATP of 50 kJ mol1 , this gives a hydrolysis
energy of PEP of about 80 kJ mol1 , although the
phosphate of PEP is formally a lousy ester. Why is this so?
The answer is carbonyl again. Pyruvate carries a carboxyl
group at C1 in conjugation with a carbonyl group at C2,
which can steal a proton from the C3 methyl group to
form the enol form of pyruvate. The enol form of pyruvate is
a high energy state of about +60 kJ mol1 , so it occurs only
in one out of 22 billion molecules. When the phosphate is
transferred from PEP to ADP, formation of the ATP takes
+50 kJ mol1 , 20 kJ mol1 are contributed from the energy
of the ester bond, and additional 60 kJ mol1 from the
following tautomerization from the enol form back to the
normal pyruvate.
S1.3 Basic Biochemistry S33
This high energy content of PEP can be used in an inge-
nious active transport system called phosphotransferase
system or group translocation. Bacterial cells may contain
many PTS systems that import sugars such as glucose.
All these PTS systems have one common component,
a kinase EI phosphorylates a small heat-stable protein
named HPr that uses PEP as energy-rich phosphate donor.
HPr-phosphate migrates to the respective sugar-specic
importer, which is always composed of three subunits or
domains of one polypeptide chain, EIIA , EIIB , and EIIC . If
one of these factors is a domain of a longer polypeptide or
its own polypeptide depends on the individual transport
system. The phosphate residue is transferred from one of the
components to the next one. EIIC is a membrane-integral
part of the PTS system and imports the sugar. During
this process the sugar is phosphorylated by the phosphate
residue, which ultimately comes from the PEP. So, in case
of glucose, G6P is the product of the uptake reaction.
These PTS systems have four tremendous advantages
over other uptake systems. First, the heat released by the
formation of G6P from PEP is about 60 kJ mol1 , leading
to a theoretical 22 billion-fold accumulation of sugar phos-
phate inside to sugar outside. If ATP is used, this energy
would be 30 kJ mol1 or the square root of 22 billion,
which is only 149 000-fold. Second, the G6P is ready
to be used and does not have to be activated rst. Third,
one molecule of glucose yields after glycolysis two PEP,
importing two glucose, yielding four PEP, importing four
glucose, yielding eight PEP, and so on: in a chain reaction,
glycolysis and PTS are coupled with care for a rapid pro-
vision of energy to the PTS systems. Fourth, PTS systems
can be used for regulation of the cellular metabolism. If,
for instance, glucose is used up, a phosphate jam results,
all PTS components are phosphorylated and phosphate
transfer stops. In this case, EIIA -phosphate migrates to an
enzyme called ATP-cyclase that forms 2 ,3 -cyclic AMP
(cAMP) from ATP. This messenger is bound by the CAP
(cyclic-AMP-acceptor protein; alternative name CRP, CRP
(catabolite-repressor protein); the resulting complex binds
to DNA and activates transcription of the genes for other
catabolic systems, for example, for degradation of lactose,
maltose, or arabinose.
PEP is also an important anabolic compound and needed,
for instance, for the lactyl-ether of muraminic acid in
the bacterial peptidoglycan cell wall (see above), or for
anaplerotic reactions of the TCA cycle (see below).
S1.3.4
Cycles and Shunts Attached to the F1,6bP-Pathway
S1.3.4.1 Overview
Some important reaction cycles operate close to the
upper part of the F1,6bP pathway and are used as alter-
native degradation (2-keto-3-deoxy-6-phospho-gluconate,
KDPG) or fermentation (phosphoketolase) shunt for
S34 S1 Basic Biochemical Roots
glucose, to synthesize pentoses and NADPH (pen-
tosephosphate cycle (PPC)), assimilate carbon dioxide
(ribulose-bis-phosphate or Calvin cycle) or acetalde-
hyde (ribulose-mono-phosphate). Moreover, metabolic
branches lead to synthesis of building blocks of the primary
metabolism (see Section S1.3.9) but also to secondary,
specialized compounds (see Chapter 2).
S1.3.4.2 KDPG Pathway
Glucose is an attractive substrate for chemoorganohetero-
trophs and thus heavily competed for. Proteobacteria
roughly belonging to the genus Pseudomonas (Gammapro-
teobacteria) are aerobic bacteria that respire with molecular
oxygen or nitrate under anaerobic conditions. This leads
to conservation of about 38 mol ATP per mol glucose. As
outlined above, homofermenting lactic acid bacteria (Lac-
tobacillales, Firmicutes) conserve only 2 mol ATP per mol
glucose fermented to 2 lactate (plus 2 protons exported),
so, to reach the same growth rate as a Pseudomonas, a lactic
acid bacterium has to metabolize glucose 19 times as fast as
the aerobic bacterium.
To deal with this tremendous consumption rate of its
competitors, pseudomonads perform a trick. They do
not import the glucose but reduce the sugar directly at
their surface to gluconic acid. The resulting electrons are
transferred via a prosthetic group, a chinon named PQQ
(pyrrolochinolinchinon or methoxatin), directly into the
respiratory electron transfer chain. By ultimate transfer to
molecular oxygen, these electrons can be used to conserve
energy (about 2 ATP per mol glucose) as rapidly as the lactic
acid bacteria. This process is used to produce the nonalco-
holic beverage Bionade from malt, sugar, water, and fruit
essences. Moreover, in a similar and also biotechnologically
important process, ethanol is oxidized to acetic acid. Bacte-
rial genera responsible for such an incomplete respiration
are Gluconobacter and Acetobacter (Alphaproteobacteria).
To deal later on with the gluconate, pseudomonads
contain a degradation pathway dierent from the F1,6bP
pathway; however, it should be noted that the F1,6bP
pathway may be needed for anabolic reactions neverthe-
less. In this KDPG pathway of pseudomonads and other
bacteria, G6P is rst oxidized to 6-phospho-gluconate
(via an intermediary 6-phospho-gluconolacton ring) in a
NAD+ or NADP+ -dependent carbonyl-reaction: water is
added to the C1 aldehyde giving a semi-acetal. From here, a
hydride can be transferred to NAD(P)+ yielding NAD(P)H,
a proton, and 6-phospho-gluconate. If external gluconate is
consumed, it has to be imported rst and phosphorylated
in an ATP-dependent kinase reaction.
In a reaction similar to the 2PG enolase, water is elim-
inated from 6-phospho-gluconate giving KDPG, the name
giver of the pathway. The rst three atoms of KDPG resem-
ble pyruvate and, thus, KDPG can split by an aldolase reac-
tion to pyruvate and 3GAP. Similar to the F1,6bP aldolase
in the reverse point of view, the CH acidic C3 of pyruvate
(instead of C1 of DAHP) attacks the C1 carbonyl of 3GAP.
Thus, the KDPG pathway transforms 1 mol of glucose up
to now into pyruvate, 3GAP, needs 1 ATP for activation,
and produces 1 NAD(P)H from NAD(P)+ . The 3GAP is
metabolized by the lower part of the F1,6bP pathway to a
second pyruvate, 2 ATP, and another NADH. In respiring
organisms, the NAD(P)H moieties are regenerated by trans-
fer of the electrons to molecular oxygen in a respiratory
chain; however, the proteobacterium Zymononas mobilis
(Alphaproteobacteria) ferments glucose to ethanol and
CO2 using the KPDG pathway. The two pyruvate molecules
are decarboxylated by the pyruvate decarboxylase. This
enzyme contains TPP (see above) that attacks with a free
electron pair from the CH acidic carbon atom of this
prosthetic group the C2 carbonyl carbon of pyruvate.
This leads to decarboxylation and release of CO2 and
hydroxyl-methyl-TPP. In a second carbonyl reaction, TPP
is regenerated and acetaldehyde CH3 -CHO released. The
C1 aldehyde can now be reduced to an alcohol by hydride
transfer from NADH in a third carbonyl reaction.
Z. mobilis conserves only 1 ATP per mol of glucose and
the former glucose atoms that become CO2 are C1 and C4
compared to C3 and C4 in yeast. This dierence can be used
to examine ecosystems in situ for the pathways running in
the organisms there by feeding isotope-labeled glucose to
the respective site.
Because gluconate already carries a negative charge, some
archaea oxidize it in a modied KDPG pathway glucose rst
to gluconate and phosphorylate the gluconate. Others even
eliminate water from gluconate to KDG and phosphorylate
this compound, or even others split KDG to pyruvate and
glycerin-aldehyde before this compound is at least phospho-
rylated.
S1.3.4.3 Heterofermentative Lactic Acid Fermentation
and Phosphoketolase
Pseudomonads oxidize glucose rapidly to gluconate to com-
pete with lactic acid bacteria. Heterofermentative lactic acid
bacteria (Lactobacillales, Firmicutes) have evolved a path-
way that allows a very ecient competition with pseudo-
mads or homofermentative lactic acid bacteria, and to fer-
ment gluconate and pentoses.
As in other bacteria, glucose is usually imported by the
PTS system (see above) that allows a highly ecient accu-
mulation as G6P. As in the KDPG pathway, G6P is oxidized
by hydride transfer to NAD(P)+ to 6-phospho-gluconate.
Do note that the free energy that results from the oxidation
of an aldehyde to a carboxyl group is not conserved as in
the case of the GAPDH reaction but completely released
as heat, driving the reaction equilibrium strongly to 6-
phospho-gluconate. In contrast to the KDPG pathway, not
elimination of water is the next step but NAD+ -dependent
oxidation of the C3 alcohol group to 3-keto-6-phospho-
gluconate KPG, which can be understood again in the
other direction as transfer of a hydride ion from NADH
to the carbonyl-C3. Following the carbonyl function at
C3, the C2 atom is CH acidic, and KPG spontaneously

decarboxylates to D-ribulose-5-P (Ribu-5-P) and CO2 ,
which drives the direction again into the pathway of the
ribulose. D-ribulose is one of the two D-keto-pentoses
with the two stereoisomeric C3 and C4 carbons in the
DD conformation; the other one is D-xylulose with LD:
D-ribulose-5-P is epimerized to D-xylulose-5-P via the enol
intermediate common to both compounds, similar to the
hexose phosphate isomerase reaction.
The next step, the xylulose-5-P phosphoketolase reaction,
uses TPP again to attack the C2 carbonyl group of xylulose-
5-P, leading to a TPP-attached pentose that splits into
3GAP and dihydroxy-ethyl-TPP (H2 COH-HCOH-TPP). In
the inverse reaction, which is realized in transketolases (see
below), the carbon adjacent to the TPP carries a negative
partial charge and can attack with the electron pair of this
bond the carbonyl-C1 of 3GAP, similar to the C1 of DHAP
in the F1,6bP aldolase reaction. In the phosphoketolase
reaction, the dihydroxy-ethyl-TPP eliminates water to
hydroxy-vinylTPP (H2C=COH-TPP). This is the enol
form of acetyl-TPP and consequently tautomerizes to this
product (CH3-CO-TPP). Because of the negative partial
charge of the carboxyl-carbon of the acetyl group, this is
an energy-rich bond. In a phosphoclastic reaction similar
to that in the 3GAP-DH, an oxygen of the phosphate
attacks the carboxyl-C of the acetyl group, leading to
acetyl-phosphate and regenerated TPP. Acetyl-phosphate
contains an energy-rich mixed acid anhydrite group and
can be used to phosphorylate ADP to ATP.
Thus, heterofermentative lactic acid bacteria ferment
glucose up to now to 3GAP, CO2 , and acetyl-phosphate at
the expense of the production of 2 NADH but in a pathway
with very ecient reactions. As in the KDPG-pathway,
3GAP follows the lower F1,6bP pathway, leading to pyru-
vate, another NADH, and 1 ATP, and as in the fermentation
pathway of the homofermentative lactic acid bacteria
(S1.3.5), this third NADH is regenerated to NAD+ by the
reduction of pyruvate to lactate (see below). So, at this point,
glucose is fermented to lactate, CO2 , acetyl-phosphate, and
still two NADH have to be regenerated. This can be done
by the reduction of acetyl-phosphate via acetaldehyde to
ethanol, so that glucose is now nally fermented to lactate,
ethanol, and CO2 , yielding only one ATP.
Only one ATP per mol of glucose would not allow a het-
erofermentative lactic acid bacterium to compete eciently
with a homofermentative lactic acid bacterium that earns
the double income; however, heterofermentative lactic acid
bacteria use external electron acceptors to regenerate some
of their NADH. Such acceptors could be fructose yielding
the sugar alcohol mannitol, or even molecular oxygen. In
contrast to respiring organisms, no electron-transporting
respiratory chain is used. Nevertheless, if the heterofer-
mentative lactic acid bacterium is able to regenerate two
NADH in such a way, the acetyl-phosphate is not needed
as electron acceptor, the phosphate group can be trans-
ferred to ADP to give ATP, and the bacterium conserves 2
ATP/mol glucose as its homofermentative competitor but
S1.3 Basic Biochemistry S35
using a much more ecient pathway. So, as long as there
are external electron acceptors such as residual molecular
oxygen or fructose, heterofermentative bacteria are the
fastest glucose fermenters.
Furthermore, they can switch to gluconate if the glucose
has been consumed. Without an external electron acceptor,
gluconate is fermented to lactate, CO2 , 0.5 acetate and 0.5
ethanol, and 1.5 ATP. As gluconate needs to be reduced
only once for entry into the phosphoketolase pathway,
only half of the acetyl-phosphate is needed as electron
acceptor compared to glucose, while the other half can
be used to conserve energy by the production of ATP.
When external electron acceptors are present, the products
from gluconate are also lactate, acetate, and CO2 as in
case of glucose. Finally, heterofermentative lactic acid
bacteria can also ferment pentoses to lactate and acetate via
xylulose-5-P yielding 2 ATP. No external electron acceptors
are needed here.
With all these tricks, heterofermentative lactic acid
bacteria are the most allochthonous organisms as far as
carbohydrates are present that can be rapidly fermented.
When neither electron acceptors nor gluconate or pentoses
are available, homofermentative lactic acid bacteria take
over, which are the second-most allochthonous organisms.
There is, however, one group of lactic acid bacteria that
are not allochthonous but in contrast evolved into the
autochthonous direction: the bidobacteria.
Bidobacterium bidum (Actinobacteria) is the predom-
inant settler of the gut of newborn human babies as long as
they are nursed with mothers milk, which contains the cell
wall building block N-acetyl-glucosamine that is essential
for this bacterium. B. bidum epimerizes G6P to F6P and
splits an F6P by a F6P-phosphoketolase to acetyl-phosphate
and the tetrose D-erythrose-4-P. As F6P looks like xylulose-
5-P with an additional hydroxyl-carbon in the D stereo
conformation in the middle and erythrose-4-P also like
3GAP with an additional HCOH in D, both TPP-dependent
reactions are very similar. A second F6P is cleaved in
an aldolase-like reaction similar to the F1,6bP aldolase;
however, in this transaldolase the DHA (dihydroacetone)
moiety remains attached to the enzyme as Schi base, is
not released but transferred instead to the erythrose-4-P
leading to 3GAP and a keto-heptose, seduheptulose-7-P.
Again, this compound looks like xylulose-5-P or F6P but
with yet another HCOH in the D stereo conformation.
Similar to the phosphoketolase reaction, TPP can be used
to attack the seduheptulose-7-P, to remove a dihydroxy-
ethyl-TPP and release ribose-5-P. In this transketolase
reaction, the dihdroxyethyl-TPP is not transformed into
acetyl-TPP but the C2 moiety is transferred to the 3GAP
that has been previously synthesized in the transaldolase
reaction, giving xylulose-5-P. The ribose-5-P is also iso-
merized to ribulose-5-P and epimerized to a second
xylulose-5-P, and in a xylulose-5-P phosphoketolase reac-
tion, both xylulose-5-P are cleaved into a 3GAP and an
acetyl-phosphate each. So, without the production of any
S36 S1 Basic Biochemical Roots
NADH up to now (!), 2 glucose have been fermented into
3 acetyl-phosphate and 2 3GAP at the expense of 2 ATP
needed for activation (or 2 PEP needed for PTS-driven
import). The two 3GAP are further processed to two lactate
similar to the other lactic acid bacteria. In total, B. bidum
ferments 2 glucose to 2 lactate, 3 acetate, and a net value of
5 ATP/2 glucose of 2.5 ATP/glucose.
Do note two important new classes of enzymes, transal-
dolases, and transketolases. Transaldolases function like
the F1,6bP aldolase but the DHA moiety remains attached
as Schi base to the enzyme and can be transferred to other
aldoses: to 3GAP giving F6P or to erythrose-4-P giving
seduheptulose-7-P. Similarly, the dhydroxy-ethyl-TPP
remains at the transketolase enzyme and can be attached to
3GAP giving xylulose-5-P, to erythrose-4-P giving F6P, or
to ribose-5-P yielding seduheptulose-7-P. Combining the
action of both enzyme types is an ecient way to create
a pentose, tetrose, and heptose pool, which is required
for many anabolic reactions such as the synthesis of
nucleotides.
S1.3.4.4 Pentosephosphate Cycle (PPC)
This is what is exactly done in the PPC. In addition to the
creation of a pentose, tetrose, and heptose pool, the cycle
produces NADPH for anabolic reactions.
Reminescent to the pathway in heterofermentative lactic
acid bacteria, G6P is oxidized to 6-phosphogluconate and
NADPH plus a proton, and in a second step to ribulose-5-P,
CO2 , and a second NADPH/H+ . Three ribulose-5-P are
epimerized to two xylulose-5-P and isomerized to one
ribose-5-P. The rst transketolase reaction removes the
two carbon atoms from xylulose-5-P as intermediary
dihydroxyethyl-TPP releasing 3GAP, and transfers it moiety
to ribose-5-P giving seduheptulose-7-P. In the transal-
dolase reaction, the terminal DHA moiety is taken from
seduheptulose-7-P releasing erythrose-4-P and fused to the
3GAP giving F6P. Finally, the second transketolase moves
the dihdroxyethyl-group from the other xylulose-5-P to the
erythrose-4-P yielding a second F6P and again 3GAP. The
cycle can be closed by the isomerization of F6P to G6P and
half of the 3GAP to DHAP, aldolase reaction to 0.5 F1,6bP,
phosphatase reaction to 0.5 F6P, and again isomerization to
0.5 G6P.
So, in total, three G6P are converted into 2.5 G6P, 3
CO2 , and 6 redox equivalents as NADPH: (<CH2 O>)3 + 3
H2 O 3 CO2 + 32[H]. Do note the ecient arrangement
of the PPC. If NADPH is needed, G6P is oxidized at the
entrance part of the PPC to pentoses, CO2 , and NADPH.
If no pentoses are needed, they can be completely recycled
to G6P again but any amount of pentoses can be taken out
for anabolic reactions. If more pentoses are needed than
NADPH, the cycle can also turn in the other direction
and produce them from F6P by the transketolase and
transaldolase reactions.
Also note the importance of TPP in these reactions
and the dierence between the dihydroxy-ethyl-TPP
intermediate of the transketolase and phosphoketolase
reactions, which is actually an energy-rich acetyl moiety
bound to TPP, and the hydroxyl-ethyl-TPP of the pyruvlate
decarboxylase, which is a bound acetaldehyde that has to
be oxidized to form an energy-rich bond (see S1.3.5).
S1.3.4.5 Calvin Cycle
With only a few modications, the most important bio-
chemical cycle for the assimilation of carbon dioxide, the
Calvin cycle (Figure S1.16), is nothing more than a PPC
turning in the F6P pentose direction. Remember that
the CO2 -releasing step of the PPC and related reactions
yields ribulose-5-P because the C3 carbonyl group of the
2-keto-6-phospho-gluconate makes the C2 and the C4
carbon atoms CH-acidic, so in the other direction, the
CH-acidic C1 and C3 atoms of ribulose-5-P may attack
a carbonyl carbon atom such as in O=C=O or carbonate.
Such a reaction, however, would be very slow with an
equilibrium in the wrong direction. To prevent this, the
C1 carbon of ribulose-5-P is blocked by the transfer of a
second phosphate in the phosphoribulo-kinase reaction,
leading to ribulose-1,5-bisphosphate (RuBP). This reaction
also draws the equilibrium toward RuBP by the release of
about 30 kJ mol1 of heat.
The enol-tautomer of RuBP serves subsequently as accep-
tor for an addition of carbonate, leading to two molecules of
3PG, the well-known intermediate of the F1,6bP pathway. In
a blind reaction, molecular oxygen O=O can also be added
to the double bond of this enol tautomer, yielding one 3PG
and phosphoglycolate, which has to be degraded to CO2 or
transformed into sugars again by long anabolic pathways.
This light respiration decreases the yield of carbon xa-
tion by the Calvin cycle but can obviously not be avoided in
aerobic or even oxygen-producing cells (Figure S1.17).
The 3PG molecules are used to recycle the acceptor
ribulose-1,5,bP again. The substrate ow moves rst in the
gluconeogenesis direction of the F16bP pathway from 3PG
via 1,3bPG (1 ATP) to 3GAP (1 NADPH). A second
3GAP is isomerized to DHAP and fused with the rst
3GAP to F1,6bP, which yields F6P in the phosphatase
reaction. Similar to the PPC, a transketolase transfers the
dihydroxyethyl-group from F6P to the third 3GAP leading
to erythrose-4-P and the rst xylulose-5-P. In contrast to the
PPC, the erythrose-4-P is not substrate of a transaldolase
reaction but of an aldolase that fuses erythrose-4-P and
3GAP number four to seduheptulose-1,7-bP, which is also
dephosphorylated to seduheptulose-7-phosphate. Both
phosphatases (F1,6bPase, Sedu1,7bPase) and the phospho-
ribulokinase reaction generate the heat that turns the cycle
in the correct orientation. In a second phosphoketolase
reaction, a dihydroxyethyl-moiety is transferred from
seduheptulose-7-phosphate to 3GAP number ve, yielding
ribose-5-P, and xylulose-5-P. All three pentose phosphates
are nally epimerized and isomerized to ribulose-5-P.
In total, carboxylation of three ribulose-1,5-bisphospate
molecules gives six 3PG. Five of them are needed to recycle

CO2 (HCO3)
3-Phosphoglycerate
(3PG)
Rubisco
Ribulose-1,5-
bisphosphate
Carboxylation
PSI
ATP
ADP
Ribulose-5-
phosphate Regeneration
Ribose-5-
phosphate
Nucleotides
Histidine
Figure S1.16 Calvin cycle. A series of light-independent enzy-
matic reactions in the stroma of chloroplasts converts CO2 and
H2 O into organic compounds, using ATP and NADPH from the
the three ribulose-1,5-bisphospate acceptors. Formation
of six 3GAP needs 6 ATP and 6 NADPH, recycling of the
acceptor additional 3 ATP. One molecule of 3GAP is the
product of the cycle and can be used to synthesize 1/2
glucose molecule. In the net reaction and calculated per
mol of glucose, 6 CO2 + 12 NADPH/H+ > C6 H12 O6 + 12
H2 O + 12 NADP+ , and 18 ATP are needed per mol of
glucose.
Some plants (C4-plants, CAM-plants) use CO2 -xing
reactions before the reaction catalyzed by the ribulose-
bisphosphate-carboxylase to increase the eciency of this
process (Figure S1.18 and S1.19).
S1.3.4.6 Ribulose-Monophosphate Cycle
The Calvin cycle needs more energy per mol of glucose
than other assimilation pathways for carbon dioxide (see
below) but seems to be able to function at comparable
low concentrations of CO2 . The energy is used for the
reduction of the carboxylic group of 3PG and the activa-
tion of ribulose-5-phosphate, and to block its C1 carbon
atom. This becomes evident if we look at the assimilation
reaction of bacteria that oxidize methane and other one-
carbon compounds. These bacteria do not need to use the
S1.3 Basic Biochemistry S37
PSI
ATP
ADP
1,3-Bisphosphoglycerate
(1,3-BPG)
Reduction
PSI
Amino
NADPH + H+ Sucrose
acids
NADP
PEP
Glyceraldehyde 3-phosphate
(3GP)
Biological
oxidation
Erythrose- Aromatic
4-phosphate amino acids
photosynthetic light reactions. The key enzyme for carbon x-
ation in this cycle is the ribulose-1,5-bisphosphate carboxylase
(PSI photosystem I) (Graphics: G.-J.Krauss, D. Dobritzsch.)
expensive Calvin cycle because methane or methanol are
oxidized to CO2 via formaldehyde H2 CO, which is toxic on
the one hand but conveniently on the redox level <CH2 O>
of the carbohydrates on the other. Thus, there is no need to
activate a carboxylic group to reduce it to an aldehyde and
this energy costs need not be provided.
There are two important pathways for the xation of
formaldehyde. The serin pathway is not outlined here.
In the ribulose-mono-phosphate cycle, ribulose-mono-
phosphate attacks with its CH-acidic C1 carbon atom the
carbonyl-carbon of formaldehyde, leading to a hexulose-6-P
compound with a C3 carbonyl-group that can easily iso-
merize via the common enol compound to F6P. Multiplied
by a factor of 3, three formaldehydes assimilated by three
ribulose-5-phosphate yield three F6P.
To regenerate the acceptors, one F6P is phosphorylated to
F1,6P at the expense of 1 ATP, and the F1,6bP cleaved into
3GAP and DHAP. This step is followed by two transketolase
reactions plus a transaldolase reaction. The rst transke-
tolase produces xylulose-5-P and erythrose-4-P from the
3GAP, the second F6P, the transaldolase seduheptulose-7-P
and 3GAP from the erythrose-4-P, and the third F6P,
the second transketolase ribose-5-P and xylulose-5-P
S38 S1 Basic Biochemical Roots

O2 CO2
Ribulose-1,5-
NH4+ bisphosphate

-Ketoglutarate Rubisco Calvin

Cycle
2-Phospho- 3-Phospho-
GS/GOGAT
glycolate glycerate
cp
Glu Pi ATP ADP
Malate Glycolate Glycerate Glycerate

Malate

Glycolate Glycerate NADH/H+ NAD+

O2 H2O2 NADH NAD+

Glyoxylate Hydroxypyruvate Hydroxypyruvate

Glu
p

Ser
-Ketoglutarate
Gly

NADH/H+
Gly Ser
mt
NAD+ NH4+ CO2

Figure S1.17 Photorespiration. Ribulose-1,5-bisphosphate-
carboxylase can also oxygenate ribulose-1,5-bisphosphate (RuBP)
that results in the incorporation of oxygen into the carboxyl groups
of 3-Phosphoglycerate and 2-phosphoglycolate. The two carbon
molecule is metabolised back to RuBP via reactions in dierent
cell compartments. Photorespiration happens when the CO2 level
inside the leaf becomes low and the O2 ratio is increased relative
to CO2 concentration. Photorespiration is crucial for carbon salvage
and adjustment of various metabolic functions. GS Glutamine
synthetase; GOGAT Glutamate -ketoglutarate (oxoglutarate)
aminotransferase; Rubisco Ribulose 1,5,bisphosphate carboxylase-
oxygenase (Graphics: G.-J. Krauss, D. Dobritzsch.)

from the seduheptulose-7-P and the 3GAP. Again, the
two xylulose-5-P and the ribose-5-P are epimerized and
isomerized to three ribulose-5-P, closing the cycle.
Net product is one molecule of DHAP, which may be used
to synthesize 1/2 glucose. One ATP/DHAP is hydrolyzed,
that is, 2 ATP/mol glucose, much cheaper than the energy
needed for the Calvin cycle!
S1.3.5
Fates of Pyruvate
Pyruvate is another hub in the metabolic pathways of the
cell, especially in the pathways of fermenting bacteria. Many
of these use the F1,6bP pathway to degrade glucose and con-
serve energy by substrate step phosphorylation in the 3GAP
dehydrogenase reaction (3GAP-DH); however, this reaction
is essentially connected to the formation of NADH. So, if
such a bacterium degrades glucose to 2 pyruvate, it is able
to conserve 2 ATP but has to deal with 2 NADH. Respiring
bacteria may transfer the electrons from these NADH to an
external electron acceptor in an electron transport chain;
however, fermenting bacteria are independent of these
external (and maybe limiting) electron acceptor. Instead,
they use an internal electron acceptor at he expense of
energy that cannot be conserved, and this internal electron
acceptor is made from pyruvate in many cases.
Homofermenting lactic acid bacteria perform the easiest
reaction. In a carbonyl-reaction, the hydride from NADH
is transferred to the partial positive C2 carbonyl-carbon of
pyruvate. A subsequent protonation yields an alcohol group
at C2 of the resulting lactate. Depending on the hydride
that comes from below or above the carbonyl group,
D-lactate or L-lactate is formed. The free energy of this step
is 25.2 kJ mol1 , leading to a 22 100-fold ratio of lactate
to pyruvate. This high concentration and the low pK a of
lactate can be used to additionally conserve some energy:
the molecule is exported as lactic acid in the protonated
form, deprotonates outside of the cell immediately and
the proton can be used to generate a pmf. Nevertheless, 2
pyruvate plus 2 NADH forms 2 lactate, and 2 NAD+ are
regenerated again. Homofermenting lactic acid bacteria use
the F1,6bP pathway for glucose degradation, produce per
 S1.3 Basic Biochemistry S39

HO COOH
Malate Malate COOH
NADP+
NADPH/H+ MDH

O COOH
COOH
Oxaloacetate
NADPH/H+
CO2 CA HCO3 PEPC ME CO2 Carboxylation Reduction
Calvin cycle
COOH NADP+
O-P Phosphoenolpyruvate
CH2
Regeneration
AMP+PPi PPDK
ATP+Pi+ COOH
Pyruvate Pyruvate O
CH3

Mesophyll cell Bundle sheath cell

Figure S1.18 C4-carbon xation. C4-plants evolved a special CO2
xation in mesophyll cells via phosphoenolpyruvate (PEP) carboxy-
lase, formation of C4 compound, usually malate, which is trans-
ported to the bundle sheet cells, where after its decarboxylation
CO2 is xed nally by ribulose-1,5-bisphophate carboxylase. C4
carbon xation: NADP+ -malic enzyme type. (CA Carboanhydrase;
MDH NADP+ -malate dehydrogenase; ME NADP+ -malic enzyme;
PEPC Phosphoenolpyruvate decarboxylase; PPDK Pyruvate
orthophosphate dikinase (Graphics: D. Dobritzsch.)

ATP
O
H+ ATPase H+ HO O

HO COOH

ADP+Pi O COOH
Malate O
H+ vac
MS
NADP+
NADPH/H+ MDH Malate

O COOH
COOH
Oxalacetate
NADP+
CO2 CA HCO3 PEPC CO2
Carboxylation Reduction NADPH/H+ ME
COOH Calvin CO2
Phosphoenolpyruvate O-P cycle
Sucrose,
CH2
starch, Regeneration
fructans PPDK
AMP+PPi

ATP+Pi+
Pyruvate
COOH
O
CH3
Night Day

Opened Stomata Closed Stomata

Figure S1.19 Crassulacean acid metabolism (CAM) is function-
ing in some plants living in arid conditions. In these plants stom-
ata open during night. Then CO2 is transformed to malate and
stored. During daytime stomata are closed to avoid loss of water
and CO2 is liberated from malate and fed into the Calvin cycle,
blue arrows reactions in the night; black arrows reactions dur-
ing day light (MDH NADP+ malate dehydrogenase, MS malate
shuttle, PEPC Phosphoenolpyruvate carboxylase, PPDK Pyruvate
orthophosphate dikinase (Graphics: D. Dobritzsch.)
S40 S1 Basic Biochemical Roots
mol of glucose 2 lactate, conserve 2 ATP, plus two proton
exported.
The eukaryotic yeast S. cerevisiae and the bacterium
Zymomonas mobilis regenerate the 2 NAD+ from the
3GAP-DH reaction by the formation of 2 ethanol and
2 CO2 . The TPP-dependent pyruvate decarboxylase
(see above) splits pyruvate into CO2 and acetaldehyde
CH3 CHO. The C1 aldehyde can be reduced to an alco-
hol by hydride transfer from NADH in a third carbonyl
reaction. Thus, yeast produces 2 ethanol and 2 CO2 from
1 mol of glucose, and 2 ATP. The ethanol is important for
the production of beer and whine, the CO2 for baking. The
pathway that Zymomonas uses (KDPG) is described above
(S1.3.4.2) and yields 2 ethanol and 2 CO2 from 1 mol of
glucose too but only 1 ATP.
Some anaerobic bacteria such as the Enterobac-
ter group of the enterobacteria (Enterobacteriaceae,
Gammaproteobacteria) also decarboxylate pyruvate during
their fermentation to hydroxyl-methyl-TPP and CO2 .
In contrast to yeast, no acetaldehyde is released but
the acetaldehyde moiety is transferred in a carbonyl-
reaction to a second pyruvate, leading to 2-acetyl-lactate,
which becomes subsequently decarboxylated to acetoin
(CH3 HCOHCOCH3 ). Enterobacter degrades glucose
also via the F1,6bP pathway and 1/3 of the NADH produced
in the 3GAP-DH reaction can be regenerated to NAD+ by
the transfer of the redox equivalents to acetoin, yielding
2,3 butandiol (CH3 HCOHHCOHCH3 ). To regenerate
the remaining 2/3 of the NADH, pyruvate is cleaved into
formiate HCOOH and the energy-rich compound acetyl-S-
CoA (pyruvate formiate lyase). Subsequently, acetyl-S-CoA
is reduced in two steps via acetaldehyde to ethanol. E. coli,
another enterobacterium, does not produce 2,3 butandiol
but contains a pyruvate formiate lyase. Enterobacteria are
facultative anaerobic bacteria and use this enzyme only
under anaerobic conditions.
Aerobic organisms usually possess a pyruvate dehydroge-
nase (PDH) complex, a complicated enzyme complex that
nevertheless starts with a TPP-dependent pyruvate decar-
boxylase reaction as described above but named here PDH.
Again, the product of this reaction is hydroxyl-methyl-TPP;
however, acetaldehyde is again not released. Instead, a sul-
fur atom attacks the hydroxyl function in the dihydrolipoyl
transacetylase. This sulfur atom is provided by an oxidized
liponamide group SS. Again, looking at the reverse acids and storage compounds, and leading to secondary,
reaction leads to some insights: here, a reduced form of
lipoamide carries an acetyl thioester CH3 COS (see Chapter 2).
at one sulfur and a hydrogen at the other (SH). The
CH-acidic carbon of TPP now attacks the carbonyl C,
leading to hydroxyl-methyl-TPP and oxidized lipoamide.
Reminiscent to the 3GAP-DH reaction, oxidation of a
formal aldehyde (the hydroxyl-methyl group attached to the
TPP) by a sulfur-dependent reaction leads to an energy-rich
bond, again a thioester. In contrast to 3GAP, however, the
direct electron acceptor for this reaction is not NAD+ but
lipoamide that becomes reduced in the process.
In the next step, the second part of the dihydrolipoyl
transacetylase reaction, the acetyl group is transferred to
HS-CoA by a carbonyl reaction; the sulfur of HS-CoA
attacks the carbonyl carbon of the acetylated lipoamide.
This leads to acetyl-S-CoA and reduced lipoamide. As the
dierence in electronegativity of S and H is small, the SH
bond is nearly covalent and carries no partial charge to allow
the transfer of a hydride ion from reduced lipoamide to
NAD+ . Thus, in the third step of the pyruvate-DH reaction,
the dihydrolipoyl dehydrogenase, the two hydrogen atoms
are rst transferred to reduced FAD and nally from here to
NAD+ . So, in the net reaction, pyruvate is decarboxylated
and oxidized to acetyl-S-CoA, CO2 , and NADH plus a
proton is formed.
Some anaerobic fermenting bacteria such as those
belonging to the genus Clostridium (Firmicutes) use the
negative redox potential of the oxidation of pyruvate to
acetyl-S-CoA to transfer the electrons not to NAD+ with a
Eo = 320 mV but to ferredoxin, a small protein contain-
ing an ironsulfur center with a redox potential of about
Eo = 390 mV. A reduced ferredoxin with such a low redox
potential can deliver its electrons to a hydrogenase, an
enzyme that reduces proton and forms molecular hydrogen
(Eo = 420 mV). This allows clostridia to remove the
redox equivalents that originated from the PDH reaction
from the balance and to create an energy-rich bond in
the acetyl-S-CoA. This energy-rich bond can be used to
conserve some energy and regenerate the NADH from the
3GAP-DH reaction.
S1.3.6
Fates of Acetyl-S-CoA
S1.3.6.1 Overview
The most important metabolic route that uses acetyl-S-CoA
in plant and other cells (Figure S1.20) is the TCA cycle
that can be used to degrade acetate completely to CO2 and
redox equivalents, to generate important building blocks for
anabolic reactions such as 2-keto-glutarate, oxaloacetate,
and succinate, or even to assimilate carbon dioxide in a
modied version of the pathway. Other pathways con-
nected to acetyl-S-CoA are those used for propionate and
butyric acid fermentation, to synthesize and degrade fatty
specialized compounds such as isoprenoids and terpenoids
S1.3.6.2 Tricarbonic Acid (TCA) Cycle
The main function of the TCA cycle (Figure S1.21) in
aerobic respiration is the complete degradation of acetate
to CO2 in the reaction CH3 COOH + 2 H2 O> 2
CO2 + 42[H]. The reaction conserves the energy-rich
bond acetyl-S-CoA, depending on the organism as ATP,
CTP, or another NTP. The four electron pairs appear as
three NAD(P)H and one electron pair on the avin level
 S1.3 Basic Biochemistry S41

Acetate Acetate
3-Phosphoglycerate 3-Phosphoglycerate Pyruvate Lipoic
acid
Pyruvate Pyruvate PDH
Cys Malonyl-CoA
ACS PDH
Malate Malate mt
Acetyl-CoA Acetyl-CoA
Sucrose
Malate
Malonyl-CoA
Amino acids CS
Succinate TCA
Fatty acids
cycle
cp Citrate Citrate
cyt
ACL

Fatty Succinate Histone

Acetyl-CoA
acids acetylation
Glyoxylate
n
-oxidation Glyoxylate
cycle Acetoacetyl-CoA Cys Protein Malonyl-CoA
Citrate acetylation
Acetyl-CoA
Isoprenoids Elongated fatty acids Flavonoids
Suberin Isoflavonoids
Acetoacetyl-CoA p Cuticle Stilbenoids
Seed oils
Isoprenoids

Figure S1.20 Fates of acetyl-CoA in plant cells. ACS acetyl-CoA synthase; ACL ATP:citrate lyase; CS citrate synthase; TCA
cycle tricarbonic acid cycle; PDH pyruvate dehydrogenase; Leu leucine; Cys cysteine; Arg arginine. (Graphics: G.-J. Krauss,
D. Dobritzsch.)

of Eo = 0 V. Overlaying this catabolic function is the pro-
duction of building blocks for anabolic reactions. As these
reactions remove constituents of the cycle, anaplerotic
reactions are needed to ll it up again, or the cycle would
be broken.
In the initial citrate synthase (CS) reaction, acetyl-CoA
attacks with its C-H-acidic methyl carbon the C2 carbonyl
atom of oxaloacetate COOHCH2 COCOOH yielding
citrate. In an iron-dependent reaction, the aconitase elim-
inates water and adds it again to the resulting double bond,
thus moving the hydroxyl-group from the C3 carbon atom of
citrate to the C2 carbon atom of isocitrate. The intermediate
cis-aconitate of this reaction carries a double bond between
C2 and C3 that is in conjugation with the carboxyl groups
in C1 and the COOH attached to C3. The rst electron
pair comes o in the isocitrate dehydrogenase reaction that
leads to NAD(P)H, H+ and oxalosuccinate that decarboxy-
lates to 2-keto-glutarate. Both are again carbonyl reactions,
the hydride transfer to the C2 carbonyl group of oxalosucci-
nate and the decarboxylation of the carboxy group attached
to C3 because the C3 atom becomes CH-acidic because of
the C2 carbonyl group.
2-keto-glutarat resembles pyruvate in the rst 3 carbon
atoms, and oxidation of 2-keto-glutarat by the 2-keto-
glutarat-dehydrogenase complex occurs similar to that
of pyruvate by its dehydrogenase complex with TPP,
lipoamide, FAD, and NAD+ as cofactors and prosthetic
groups. Products are succinyl-S-CoA, the second CO2 ,
and NADH/H+ carrying the second electron pair from
the former acetate. The energy-rich thioester group in
succinyl-S-CoA can be used in the succinat thiokinase to
create one ATP or any other NTP.
Because the succinate/fumarate couple has a redox
potential of Eo = +30 mV, the electrons from the oxida-
tion of succinate cannot be transferred to NAD+ but go
to ubichinon (+60 mV) or metachinon (+120 mV) by a
membrane-bound succinate-dehydrogenase, the complex
II of the respiratory chain of mitochondria. The fumarase
adds water to the trans-double bond of fumarate, giving
L-malate. In the nal step, the C2 hydroxy group of malate is
oxidized to the acceptor oxaloacetate and the last electron
pair is again provided as NADH/H+ .
Anaplerotic reactions ll up the TCA cycle again when
constituents are removed as building blocks. Some reactions
S42 S1 Basic Biochemical Roots

COOH CoA
S
H 2O
Pyruvate PDH O Acetyl-CoA
CH3 CH3 CS
Oxaloacetate Citrate
O COOH COOH
COOH
CoA COOH
HO
COOH
NAD+ ACO
MDH NADH/H+

HO COOH COOH
Malate COOH COOH Isocitrate
HO COOH

NADH/H+ NAD+
FUM HCO3 IDH
H2O COOH
COOH
Fumarate
COOH
FADH2
O COOH
-Ketoglutarate

Q NADH/H+
IV III I NAD+
SDH II HCO3 KGDH
COOH
Respiratory chain COOH CoA
COOH O S-CoA
FAD GTP
Succinate Succinyl-CoA
SUC
CoA
GDP

Figure S1.21 Tricarboxylic acid cycle (TCA cycle). PDH Pyruvate dehydrogenase; SUC Succinyl-Coa-synthethase; SDH Succinate
dehydrogenase; CS Citrate synthase; ACO Aconitase; dehydrogenase; FUM Fumarate hydratase; MDH Malate
IDH Isocitrate dehydrogenase; KGDH -Ketoglurate dehydrogenase. (Graphics: D. Dobritzsch.)

form oxaloacetate from PEP or pyruvate, or malate from lac-
tate. In another reaction pair, isocitrate is cleaved into suc-
cinate and glyoxylate, which is fused to an acetyl-S-CoA to
yield malate. Thus, isocitrate and acetyl-S-CoA are used to
form succinate and malate, which doubles the number of
the TCA cycle intermediates. Some organisms possess an
open TCA cycle used only for anabolism. Here, the 2-keto-
glutarate dehydrogenase is missing and the two arms of the
former TCA cycle end at 2-keto-glutarate on the one hand
and succinate on the other, giving the 2-keto-glutarate dehy-
drogenase the role to control if the TCA cycle is a catabolic
cycle or two composed of two anabolic shunts. Moreover,
nitrogen assimilation starts with 2-keto-glutarate, so, this
intermediate serves as triple switch between conservation
of energy and provision of carbon-containing and nitrogen-
containing building blocks.
S1.3.6.3 Inverse TCA cycle
To turn the TCA cycle in the opposite direction to assimilate
CO2 needs three modications. First, a fumarate reductase
is needed instead of a succinate dehydrogenase; however,
both membrane-bound enzyme perform essentially the
same reaction although in dierent directions. Second, the
2-keto-glutarate dehydrogenase is not able to x carbon
dioxide eciently. Again similar to pyruvate, the redox
potential of 2-keto-glutarate is very negative so that it is
dicult to perform the inverted reaction with NADH as
electron donor. Using reduced ferredoxin, however, with
Eo of about 390 mV makes a formation of 2-keto-glutarate
from succinyl-CoA and CO2 possible. In the third altered
reaction, citrate has to be cleaved by an ATP-dependent
enzyme, the ATP-citrate lyase, named for the reaction
in the opposite direction: citrate + ATP + HS-CoA
oxaloacetate + Ac-S-CoA + ADP + phosphate. Thus, two
ATP are needed to assimilate 2 CO2 into an acetyl-S-CoA.
To compare this reaction to the Calvin cycle, glucose has
to be produced from the acetyl-S-CoA. First, pyruvate is
synthesized by a pyruvate-ferredoxin-oxidoreductase and
CO2 , comparable to the formation of 2-keto-glutarate. For
further synthesis of glucose from 2 pyruvate, 4 ATP are
needed, so in total 8 ATP per mol of glucose compared to
18 in the Calvin cycle. Furthermore, reduced ferredoxin is
needed, which allows the reverse TCA cycle only to operate
in anaerobic organisms.
S1.3.6.4 Propionate Fermentations
Some fermenting bacteria use the TCA cycle reactions
similar to anaplerotic reactions to ferment the end products
of other bacteria. Remember that the lactic acid bacteria
produce lactate that has a low pK a value and thus lower the
pH value of their environment. Other anaerobic bacteria
such as clostridia cannot grow because their fermentation
end product, butyrate, would be protonated as butyric acid
at such a low pH value, diuse back into the cell, release

a proton in the cytoplasm, and is exported back out again
as butyrate. This cycle eciently uncouples the pmf of
clostridia, preventing their growth. On the other hand,
clostridia are ecient degraders of polymer, including
proteins. This inhibition of clostridia-mediated polymer
degradation by lactic acid bacteria is used to preserve food,
for example, by making yoghurt from milk.
Therefore, an environment containing carbohydrates
that can be rapidly fermented is rst settled by lactic
acid bacteria. For further degradation of all the other
substrates in such an environment, the environment must
either become oxic, or the lactate has to be removed.
Some clostridia are able to ferment lactate to propionate
via acrylyl-S-CoA in a very easy pathway. First, lactate is
activated by transfer to CoA that comes from the end of the
reaction pathway, from the propionyl-S-CoA. So, lactate +
propionyl-S-CoA> lactyl-S-CoA + propionate, the end
product. Second, water is eliminated from lactyl-S-CoA
forming acrylyl-S-CoA, CH2 =CH-CO-S-CoA. Third, a
hydride from NADH and a proton are added to the double
bond giving propionyl-S-CoA.
These reactions do not conserve any energy but regen-
erate NAD+ . For one molecule of propionate formed from
lactate, another lactate can be oxidized to pyruvate, which is
used in the pyruvate:ferredoxin : oxidoreductase and hydro-
genase reaction to yield H2 , CO2 , and acetyl-S-CoA, which
can be used to create an ATP. Thus, two lactate can be fer-
mented by the acylyl-CoA pathway to propionate, acetate,
H2 and CO2 , producing 1 ATP/2 lactate or 0.5 ATP per one
lactate.
A second pathway to propionate, the methyl-malonyl-
CoA pathway of Propionibacterium (Actinobacteria) is able
to conserve twice as much energy, 1 ATP per one lactate.
Lactate is again oxidized to pyruvate, giving 1 NADH that
is regenerated two steps further below. The pyruvate is
carboxylated to oxaloacetate by a biotin-mediated transfer
of CO2 from methy-malonyl-S-CoA that is also produced
further below in this shunt. Now, the reaction follows
the TCA cycle in the inverse reaction. First, the NAD+ is
regenerated by the reduction of oxaloacetate to malate.
Water is eliminated to give fumarate. In a fumarate reduc-
tase reaction, fumarate is reduced to succinate. Succinate
is activated by a CoA-transferase as mentioned above
using propionyl-S-CoA as CoA-donor. In a B12-dependent
reaction, the C4 carboxylic group of succinate is moved one
carbon atom up from C3 to C2 leading to methyl-malonyl-
S-CoA. This is the CO2 donor of the biotin-dependent
pyruvate-carboxylase, which releases propionyl-S-CoA,
which itself is the CoA donor for succinate.
The trick in this pathway is the fumarate reductase.
As the fumarate/succinate couple has an Eo = +30 mV,
the electrons for the fumarate come from the chinon
pool. Using NADH and a NADH-oxidase in a hidden
respiration, electron transport by this enzyme complex,
which is complex I in the mitochondria, from NADH to
ubichinon expels three protons, which can be used to
S1.3 Basic Biochemistry S43
synthesize 1 ATP. If the pathway from lactate to propionate
runs twice, 2 NADH can be regenerated and 2 ATP are
produced. These two NADH come from the oxidation of
a third lactate to pyruvate, and from here further on by a
pyruvate-dehydrogenase complex to CO2 and acetyl-CoA,
which can be used to synthesize another ATP. Thus, 3
lactate are fermented to 2 propionate, 1 acetate, and 1 CO2
with a net energy conservation of 3 ATP/3 lactate or 1
ATP/lactate.
S1.3.6.5 Butyric Acid and Butanol/Acetone Fermentation
Bacteria belonging to the genus Clostridium (Firmi-
cutes) or related bacteria degrade glucose via the
F1,6bP pathway to pyruvate and further on by the
pyruvate:ferredoxin:oxidoreductase and a hydrogenase
to acetyl-S-CoA, 2 CO2 , and 2 H2 , with 2 NADH left to
be regenerated and 2 ATP conserved. To deal with the
two NADH, the two acetyl-S-CoAs are fused to butyrate.
First, the CH-acidic C2 of one acetyl-S-CoA attacks the
carbonyl-C1 of a second one, forming aceto-acetyl-S-CoA
and releasing a HS-CoA. The C3 carbonyl group can be
reduced to an alcohol group to regenerate the rst NADH,
leading to 3-hydroxy-butyryl-S-CoA. Elimination of water
generates crotonyl-S-CoA that contains a C=C double bond
in conjugation with the C1 carbonyl group. The second
NADH is regenerated by the reduction of crotonyl-S-CoA
in a avin-dependent reaction to butyryl-S-CoA. So, both
NADH have been regenerated at this stage but one energy-
rich thioester bond can still be used to conserved energy.
This is done in three steps: (i) the S-CoA is transferred from
butyryl-S-CoA to acetyl-CoA; (ii) the thioester is cleaved
in a phosphoclastic reaction yielding acetyl-phosphate, a
mixed acid anhydrite; and (iii) the phosphate residue is
transferred to ADP releasing ATP and the acetate again.
So, in total, 3 ATP can be conserved by the fermentation of
glucose to butyrate, 2 CO2 , and 2 H2 .
As clostridia cannot stand low pH values because of
the uncoupling eect of butyrate, they have to stop the
production of this acid if the environment is not able to
provide sucient buering capacity and the pH value
decreases. In this case, butyryl-S-CoA, which is in fact
an activated carboxyl compound, is reduced in two
NADH-dependent steps to 1-butanol. For one 1-butanol
produced, a total of 4 NADH can be regenerated. As
the F1,6bP pathway produces only 2 NADH per glucose,
fermentation of one glucose to 1-butanol, 2 CO2 , and 2
H2 regenerates also to 2 NADH from the fermentation of
a second glucose to aceto-acetyl-S-CoA, 2 CO2 , and 2 H2 .
Thus, this energy-rich bond can be used to conserve energy
as ATP, again via acetyl-S-CoA and acetyl-phosphate. The
resulting aceto-acetate CH3 COCH2 COOH carries a
CH-acidic C2 carbon atom because of the carbonyl-C3
and can easily decarboxylate to acetone CH3 COCH3 . In
total, 2 glucose were fermented to 5 CO2 , 4 H2 , 1-butanol
and acetone, yielding 5 ATP/2 glucose or 2.5 ATP/glucose.
S44 S1 Basic Biochemical Roots
Clostridia are able to degrade polymers such as starch and
cellulose. They are also able to ferment amino acids coming
from protein degradation; however, amino acids are usually
fermented in pairs (Stickland reaction) with one amino
acid oxidized and the second reduced. In syntrophic part-
nerships (see below), clostridia are able to regenerate most
of the NADH by the production of molecular hydrogen via
ferredoxin. In such a physiological symbiosis, all kinds of
organic substances are fermented by clostridia to acetate,
H2 and CO2 , including fatty acids and previously pro-
duced butyrate, propionate, 1-butanol, ethanol, or acetone.
Butyrate, for instance, is imported again under these condi-
tions, activated to butyryl-S-CoA by the transfer of S-CoA
from acetyl-S-CoA, and oxidized in the inverse butyric acid
fermentation pathway to two acetyl-S-CoA. One of these
is used for the activation of the next imported butyrate,
the other one for the conservation of 1 ATP via acetyl-
phosphate. Prerequisition of this fermentation of 1 butyrate
to 2 acetate and 2 H2 , however, is the consumption of the
molecular hydrogen by the syntrophic partner organism.
S1.3.6.6 Fatty Acid and PHB Metabolism
Synthesis and degradation of fatty acids is not very dierent
from the production and degradation pathway of butyric
acids by clostridia. In the synthesis direction, however,
there is a dierence that allows the synthesis cycle to run
eciently into the synthesis direction. First, the enzymes
performing the synthesis are arranged in a multienzyme
complex, the fatty acid synthetase complex. Not HS-CoA,
but a part of this cofactor attached to the small acidic
protein ACP, carries the growing acyl chain. After the
rst acetyl moiety is transferred from acetyl-S-CoA to
HS-ACP yielding acetyl-S-ACP (ACP-acyltransferase), the
other acetyl moieties are rst carboxylated to malonyl-
S-CoA (COOH-CH2 -CO-S-CoA) in a two-step reaction:
ATP-dependent carboxylation of the biotin in a biotin
carrier protein followed by the transfer to the C2 atom of
acetyl-S-CoA. The rst acetyl moiety is transferred from
acetyl-S-ACP to a cysteine residue of the 3-ketoacyl-ACP
synthase so that the HS-ACP is free to accept the malonyl
moiety. In the 3-ketoacyl-ACP synthase reaction, the CH
acidic C2 atom of the malonyl-S-ACP attacks the acetyl
moiety that is in a thioester bond with the cysteine residue,
3-keto-butyryl-S-ACP is formed, and CO2 released. Note
that CO2 is not assimilated but rather has a catalytic
role. Similar to the butyric acid fermentation but with
ACP instead of CoA, the 3-keto function is reduced to an
alcohol, water is eliminated, and the resulting C=C double
bond reduced to a CC single bond. The butyryl-S-ACP is
again moved to the cysteine residue and the ACP ready for
the transfer of the next malonyl moiety. This process runs
in a cycle until the desired chain length of the fatty acid is
reached, for instance, in palmityl-S-ACP, a C16 fatty acid.
Using the acyltransferase again, the nished acyl group is
nally released as acyl-S-CoA.
Thus, hydrolysis of one ATP per acetyl moiety attached
plus the decarboxylation reaction during chain elongation
releases the heat that drives the reaction into the desired
reaction. In contrast, degradation of fatty acids in the
-oxidation is again coupled to HS-CoA and the same
reaction sequence as the degradation of butyric acid, which
is, of course, a short fatty acid.
While mammals store fat in fat cells and this fat is com-
posed of acyl groups attached to glycerol, some bacteria
have a dierent storage material, poly-3-hydroxybutyric
acid PHB. During the synthesis of this material, a pathway
similar to the butyric acid fermentation pathway is used to
form 3-hydroxybutyryl-S-CoA from two acetyl-S-CoAs. In
the following condensation step, the OH group at the C3
atom attacks the C1 carbonyl, an ester bond is formed and
HS-CoA released. As this fat-like material stores not only
carbon-bound energy but also redox equivalents, it is pre-
dominantly used in bacteria that are able to respire, while
all kind of bacteria may store glycogen. PHB can be used as
a biodegradable plastic material, which can be produced by
bacteria and transgenic plants. Moreover, other hydroxyl-
acids can be used instead of poly-3-hydroxybutyric acid,
which changes the abilities of the resulting bioplastic
material.
S1.3.7
Putting It Together: Anaerobic Ecosystems
Molecular oxygen has a low solubility in water, at a maxi-
mum 246 M at 30 C and 21%(v/v) O2 in the correspond-
ing atmosphere, but large amounts of molecular oxygen are
needed as electron acceptors in the aerobic degradation of
organic substances, for instance, 6 O2 per mol glucose. If
molecular oxygen is not produced in a site by oxygenic pho-
tosynthesis or mixed into a system by turbulences, molec-
ular oxygen is rapidly depleted. Many bacteria are able to
compensate decreasing O2 concentrations by changing their
respiratory chain to use these low concentrations eciently.
This can be done (i) by using a chinon-dependent termi-
nal oxidase instead of a cytochrome c-dependent one; (ii)
shifting from a low anity (but high turnover number) oxi-
dase to a slower one with higher oxygen anity; or (iii) by
substituting a proton-pumping NADH oxidase with a non-
pumping enzyme. Of course, the prize for these measures
to use low oxygen concentrations is a decrease in energy
conservation per mol oxygen reduced to water. Moreover,
this leads to a rapid consumption of residual oxygen at this
site, and if the concentration of organic substances is high
enough, an anoxic zone moves from a carbon-rich site in a
growing sphere to the outside. The border between the oxic
and anoxic zone is called an oxic-anoxic transition zone.
At this stage, some usually oxygen-respiring bacteria are
able to use oxidized nitrogen compounds as electron accep-
tors. Nitrate NO3 can be reduced via nitrite NO2 , NO, and
N2 O to molecular nitrogen N2 (denitrication) or via nitrite

to ammonium NH4 + (nitrate-ammonication). If these oxi-
dized nitrogen compounds are present, a second sphere fol-
lows the OATZ sphere. Inside this sphere, the concentration
of molecular oxygen decreases even further because of the
remaining microaerobic respiration processes.
If oxidized nitrogen compounds are also depleted, bac-
teria are able to grow that use sulfate as electron acceptor,
producing H2 S. H2 S reacts spontaneously but slowly with
the remaining oxygen traces, making the site completely
anoxic, and a third sphere moves away from the carbon-rich
site, closely following the OATZ and nitrate-respiration
zone. Likewise, some bacteria are able to use other oxidized
compounds such as Fe(III), Mn(IV), chromate, arsenate,
succinate, or even poly-halogenated compounds as elec-
tron acceptors under anoxic conditions until even these
acceptors are depleted. In this case, only CO2 remains as
the last electron acceptor left. Carbonate-respiring bacteria
produce acetate while archaea reduce CO2 to methane.
So, the OATZ separates an aerobic zone with high
concentrations of organic carbon from the oxic zone. In
some environments such as our gut, the enteric bacte-
ria care for an anoxic environment inside the gut while
our gut cells are aerobic. With respect to oxygen, these
bacteria are grey-zone organisms. As facultative anaer-
obic beings, they are able to respire with all kinds of
oxygen concentrations down to very low concentrations.
When the concentration of molecular oxygen becomes
too low, they switch to nitrate-ammonication. If nitrate
is gone, they respire with other electron acceptors such
as fumarate, dimethyl-sulfoxide, or trimethylamin-N-
oxide. If these acceptors are not available, they are able
to conserve sucient energy by fermentation. In this
capacity, enterobacteria serve as oxygen-buer and keep
molecular oxygen diusing out from the gut cells to reach
strictly anaerobic gut bacteria such as Bacteroides species
(Bacteroidetes/Chlorobi) .
In parallel to this transition of carbon-rich ecosystems
from aerobic to anaerobic respiration, fermentation starts.
In an environment rich in simple carbohydrates, these are
fermented to lactate, ethanol, acetate, CO2 by aero-tolerant
lactic acid bacteria, and the low pH value keeps other
bacteria from degrading the other fermentation substrates
in this site. If the oxygen concentration is suciently
low, the propionate-producing fermenters produce their
substrate from lactate, raising the pH value again. This
and nal elimination of oxygen allows the growth of the
clostridia, which degrade polymers and produce butyric
acid, butanol, acetone, and most important CO2 and
H2 . The molecular hydrogen is used by all kinds of anaero-
bically respiring bacteria so that the concentration of this
solved gas is strongly decreased. This allows the clostridia
to produce H2 even from NADH, to enter a syntrophic
partnership, and to ferment all organic substances in their
environment to acetate, CO2 and H2 .
After all other electron acceptors for anaerobic respi-
ration processes have been used, CO2 remains, which is
S1.3 Basic Biochemistry S45
produced during fermentation. Methanogenic archaea
(Euryarchaeota) reduce CO2 with molecular hydrogen
to methane, CH4 , which is a chemolithoautotrophic way
of life. These archaea are the nal syntrophic symbionts
of the clostridia. In a complicated pathway that involves
several unique cofactors, CO2 is reduced stepwisely to a
methyl group attached to tetrahydromethanopterin, which
is chemically similar to the cofactor tetrahydropterin THF.
From here the methyl group is transferred to HS-CoM
(HS-CH2 CH2 SO3 ). In the nal step close to the nickel
atom of the nickel-containing tetrapyrrole cofactor F430,
the methyl group of CH3 -S-CoM is reduced by a second
thiol compound, HS-CoB (7-mercapto-heptanoylthreonine
phosphate) to methane, and a heterodisulde CoM-S-
S-CoB is formed. Reduction of this heterodisulde by
molecular hydrogen yields Go = 40 kJ mol1 , which is
conserved in the form of pmf.
If an environment stays completely anoxic, syntrophic
partners are able to degrade all kinds of organic substances
to acetate, CO2 and CH4 . In the last step, even acetate
is cleaved by some euryarchaea such as Methanosarcina
barkeri to CH4 and CO2 . After ATP-dependent activation
of the substrate to acetyl-S-CoA via acetyl-phosphate, the
acetyl-S-CoA is cleaved in the enzyme acetyl-CoA synthase
(ACS) to a methyl moiety and enzyme-bound carbon
monoxide CO, which is oxidized to CO2 . The methyl group
is transferred to tetrahydromethanopterin and used to
conserve energy through the formation of methane as
described above. The free energy of this degradation of
acetate to CO2 and CH4 is meager, Go = 36 kJ mol1
CH4 compared to methanogenesis from CO2 , and 4
H2 of 131 kJ mol1 CH4 ; however, acetate is the nal
product of most syntrophic clostridia and degradation of
this substance becomes attractive with increasing acetate
concentrations in an environment.
The ACS is also used to assimilate CO2 in methanogenic
archaea growing on CO2 and H2 . In can also be used by
acetate-degrading sulfate-respiring bacteria and homoace-
togenic clostridia, which ferment 1 glucose to 3 acetate.
These bacteria degrade glucose via F1,6bP pathway and
pyruvate:ferredoxin:oxidoreductase to 2 acetate, 2 CO2 , 2
NADH, and 2 H2 -equivalents as reduced ferredoxin. In a
kind of syntrophic partnership alone, one CO2 is stepwise
reduced to a methyl group and the other reduced to CO by
the ACS, which nally combines the CO, HS-CoA, and the
methyl group to acetyl-S-CoA. This yields the third acetate
plus an additional ATP. This reaction seems to be smart but
homoacetogenic clostridia are not able to compete with a
syntrophic pair of partners because the energy yield from
methane formation is higher compared to the generation of
acetate.
Thus, strictly anoxic carbon-rich sites convert organic
material to CO2 and CH4 . When such a site is established,
anaerobic respiring organisms other than methanogenic
archaea produce reduced compounds such as H2 S, which
S46 S1 Basic Biochemical Roots
diuse away from this site. At the OATZ, reduced com-
pounds, fermentation end products, CH4 and H2 from one
side of the OATZ, meet molecular oxygen from the other.
Chemolithoautotrophic and microaerophilic organisms
live close to the OATZ and make a living by oxygen- or
nitrate-dependent oxidation of all these compounds. By
doing so, they serve as oxygen buer reminiscent to the
enterobacteria in the gut.
Note that a network of anaerobic and chemolithoau-
totrophic bacteria are as capable as oxygen-respiring
bacteria in degrading glucose and other organic com-
pounds completely to CO2 . As the molecular oxygen does
not come to the organic substrate, the redox equivalents of
the glucose are transferred stepwise to the oxygen, and a
variety of physiologically dierent organisms conserve their
energy with each step.
S1.3.8
Assimilation of the 10 Macrobioelements
S1.3.8.1 Carbon, Hydrogen, and Oxygen
Heterotrophic organism degrade all kinds of organic sub-
stances and feed them into the central metabolism that is
composed of the F1,6bP-backbone and the attached cycles.
From here biochemical transformation may proceed further
to CO2 or acetate, to reduced fermentation products, or
to the building blocks for macromolecules. About 50% of
the cellular dry mass are proteins, which are hydrolyzed
into small peptides and single amino acids at the end,
which enter the F1,6bP-pathway and the TCA cycle after
decarboxylation or deamination. The 20% nucleic acids
yield ribose of 2 desoxy-ribose as substrates for the PPC
and the purine or pyrimidine bases, which are degraded to
ammonium and TCA cycle intermediates. The 20% carbo-
hydrates are hydrolyzed into monosaccharides and enter
the metabolism at the upper part of the F1,6bP-pathway
or the PPC. Lipids, the remaining 10%, are composed of
glycerol that enters the F1,6bP-pathway via 3GAP and fatty
acids, which are degraded to acetyl-S-CoA units.
Autotrophic organisms are able to assimilate CO2 by
half a dozen pathways. The Calvin cycle was described in
Section S1.3.4.5, the reverse TCA cycle in Section S1.3.6.3,
and the ACS in Section S1.3.7. Other pathways such as
the hydroxyl-propionate pathway are not outlined here.
C1 compounds other than CO2 can be assimilated by the
ribulose-monophosphate pathway (Section S1.3.4.6) or the
serine pathway, which is also not further described.
All these pathways serve to assimilate the 50% carbon in
the dry mass of most cells. The 8% hydrogen also come from
the organic compounds in chemoorganohetetrotrophs, or
from reduced inorganic compounds in lithoautotrophs.
Oxygen (20%) comes mostly from organic compounds or
water. Only very few biochemical reactions deal directly
with molecular oxygen: (i) reduction of O2 to water in the
terminal oxidases of respiratory chains; (ii) detoxication
of reactive oxygen species such as hydrogen peroxide or the
superoxide radical by catalases, peroxidases, superoxide
dismutases, or superoxide reductases, respectively; and (iii)
mono- and dioxygenases that oxidase organic compounds,
mostly aromatic compounds but also CH4 and NH3 , by
direct attack with O2 .
S1.3.8.2 Nitrogen
The central portal for the 14% nitrogen is glutamate and
glutamine. At high cellular ammonium concentrations,
nitrogen can be directly assimilated into glutamate by
the glutamate dehydrogenase using 2-keto-glutarate,
NH3 , NAD(P)H, and a proton as substrates. Ammonium
attacks with its free electron pair the C2 carbonyl of
2-keto-glutarate yielding 2-imino-glutarate and water. The
carbonyl-analogous 2-imino-group can be easily reduced
by hydride-transfer to the C2 atom and a proton neutralizes
the transient negative charge of the nitrogen atom.
If the ammonium concentration is too low to allow
such a cheap assimilation of nitrogen, ATP is needed
to release some heat and to drive the reaction toward
nitrogen assimilation. This occurs in two steps. First,
in the glutamine synthetase, the C5 carboxy group of
glutamate is activated by the transfer of the -phosphate
of ATP, leading to the mixed acid anhydrite 5-glutamyl-
phosphate and ADP. NH3 can now attack the C5 carbonyl
function, leading to the release of phosphate and a C5
amide bond, which has a much lower energy (about
30 kJ mol1 ) than the mixed acid anhydrite: glutamine. In
the second step, an enzyme named GOGAT (glutamine-
oxaloglutarate-aminotransferase) creates an equilibrium
between glutamate and glutamine by the transfer of the
C5 amide nitrogen of glutamine to the C2 carbonyl of 2-
keto-glutarate (also named 2-oxoglutarate), followed by the
reduction of the resulting imino group by NAD(P)H, yield-
ing two molecules of glutamate. So, glutamine synthetase
and GOGAT together catalyze the same net reaction as
glutamate dehydrogenase but one ATP is additionally
hydrolyzed.
Some other amino acids can also be produced directly
from ammoniac. Similar to the glutamate dehydrogenase,
the aspartate dehydrogenase and the alanine dehydroge-
nase produce their products from oxaloacetate or pyruvate,
respectively. Reminiscent to the glutamine synthetase, an
asparagine synthetase may produce asparagine. Despite
these exceptions glutamate is the most important nitro-
gen donor for biosynthesis of most other amino acids in
transaminase reactions. Transamimases contain pyridoxal-
phosphate, which receives the amino-group of one amino
acid (such as glutamate in the anabolic direction) via the
formation of a Schis base, releases a 2-keto acid, and
uses the enzyme-bound pyridoxamine to transfer nitrogen
to another 2-keto acid in a classical ping-pong reaction.
This gives the glutamate/glutamine pair and especially the
glutamine synthetase the main function in the regulation
of nitrogen assimilation. Depending on the particular
organism, glutamine synthetase is a huge multisubunit

enzyme complex with a size between 350 and 600 kDa.
The individual subunits can be chemically modied by
adenylation, which changes the reactivity of the total
enzyme complex. By chemical modication, the overall
activity may be thus stepwise altered from very low to very
high, resulting in a netuning of nitrogen assimilation at
this portal in addition to e rough-tuning by the control
of gene expression. The signals that are recognized and
processed to perform this control are the concentrations
of 2-keto-glutarate (sucient carbon), ATP (sucient
energy), and glutamine (sucient nitrogen).
Heterotrophic organisms that degrade organic substances
may nd sucient nitrogen in their substrates; however,
heterotrophs living on low-nitrogen compounds or
autotrophic organisms rely on inorganic nitrogen sources.
Ammoniac is electron donor for chemolithoautotrophic
nitriers that reduce ammoniac rst to nitirite and then
to nitrate. So, in aerobic environments, the ammonium
concentration may be too low to allow nitrogen assimilation
from this source. In this case, many organisms, bacteria
as well as plants, are able to reduce nitrate by assimilatory
nitrate reduction to ammoniac again. The required nitrate
reductases contain a molybdenium cofactor (MoCo) for
this reaction.
As explained in Section S1.3.7, nitrate may be used
as electron acceptor in an anaerobic respiration, leading
to the production of nitrite and further on to NO, N2 O,
and molecular nitrogen. So, by the action of aerobic
chemolithoautotrophic ammonium oxidizers and faculta-
tive anaerobic nitrate-respiring organisms, an environment
may lose all of its bound nitrogen. It is exactly this reaction
that is used to remove bound nitrogen in sewage-treatment
plants. Nevertheless, this process poses a problem in
natural environments. It can be solved by the evolution
of nitrogen-xing bacteria and archaea (see also Section
S5.2.2). These contain the enzyme nitrogenase that is able to
reduce molecular nitrogen to ammoniac at the expense of a
lot of energy (about 16 ATP). Nitrogenases, which contain
a Mo in a cofactor completely dierent from the MoCo, are
extremely oxygen-sensitive enzymes and are produced by
the cell only in times of severe nitrogen starvation.
S1.3.8.3 Sulfur
As H2 S is very toxic, the about 1% S in biomass is acquired
mainly from organic sulfur sources or sulfate. Sulfate is
imported into bacterial cells by rather unspecic secondary
sulfate proton symporters or by primary, ATP-dependent
ABC (ATP-binding cassette) sulfate uptake systems. These
use a specic sulfate-binding protein to sequester the
anion and drag it to the importer. Binding proteins of ABC
importers are located in the periplasm of Gram-negative
bacteria or attached to the outside of the cytoplasmic
membrane in Gram-positive bacteria. So, dependent on
the sulfate concentration outside, sulfate uptake can be
a cheap process that needs only two protons of the
S1.3 Basic Biochemistry S47
Z. pH portion of the pmf (high sulfate concentrations) or
hydrolysis of an ATP (low concentrations).
As the redox potential of sulfate is too negative
(Eo = 600 mV) to allow direct reduction to sulte,
sulfate is rst activated to APS by the ATP sulfurylase.
In APS, the sulfate substitutes the -phosphate of ATP,
leading to the release of pyrophosphate, which may be
hydrolyzed to drive the reaction further into direction of
the APS. In many organisms, even a third phosphate acid
anhydrite bond is spent: APS is phosphorylated to PAPS (3
phosphoadenosie-5 -phosphosulfate).
Either APS or PAPS are reduced to sulte (SO3 2 ).
First, the sulfate group is transferred to a thiol group, for
example, of a small protein such as thioredoxine, leading to
an SSO3 -intermediate, which is an energy-rich thioester.
Attack of a second thiol group releases the sulte and
forms a disulde, which has to be reduced again. The
sulte is further reduced to sulde by an enzyme complex
containing a siroheme prosthetic group, an iron-containing
heme in which two of the pyrrole rings are reduced. Sulde
is immediately bound to O-acetyl-serine to yield cysteine.
O-acetyl-serine is an activated form of serine created by the
transfer of an acetyl moiety from acetyl-S-CoA. Cysteine
can be the sulfur donor for methionine and synthesis of
iron-sulfur centers. The APS or PAPS reductase and sulde
assimilation by O-acetyl-serine are the most important
regulatory sites for sulfur assimilation.
Some bacteria are also able to use thiosulfate as sulfur
substrate. Thiosulfate reacts with O-acetyl-serine to an
S-sulfonate derivative, which is subsequently reduced to
cysteine.
S1.3.8.4 Phosphate
Reminiscent to sulfate, phosphate is imported by fast
and unspecic importers of the phosphate inorganic
transport (PiT) protein family or an ABC-import system.
PiT proteins such as PitA from E. coli import phosphate
as metal-phosphate complexes, so they import not only
phosphate but also essential metal cations, for example,
Mg2+ . Phosphate need not be reduced or modied to fulll
its function in metabolism, it is ready to go right after
uptake.
As about 3% phosphate is needed for dry mass and the
environmental phosphate concentration may be low from
time to time, cells have developed a storage system. If
surplus, phosphate can be polymerized to polyphosphate,
which contains the phosphate moieties in acid anhydrite
bonds. Thus, polyphosphate serves also as energy-storage
compound, and this may be an ancient form of such a stor-
age system. Polyphosphate is negatively charged and stored
together with neutralizing metal cations in acidocalcisomes
and cellular organelles found in bacteria and eukaryotes.
Most organisms are able to use organic phosphate com-
pound as phosphate source. Moreover, some organisms are
able to reduce phosphite for this purpose.
S48 S1 Basic Biochemical Roots

S1.3.8.5 Metals
Of the four metals that are major bioelements, Na+ , K+ ,
Mg2+ , and Ca2+ , usually potassium and magnesium stay
in the cytoplasm while sodium and calcium stay out.
Potassium is taken up by a variety of secondary and primary
import systems. Potassium is needed to neutralize the
(mainly) negative charges of the cellular carbon compounds
and is used to control the correct osmotic pressure of the
cytoplasm. If the osmotic pressure in the environment
changes, the cell may suer water inux (pressure in
the cytoplasm higher than outside) or eux (vice versa).
Rapid export of K+ decreases the osmotic pressure in the
cytoplasm to deal with the rst problem and K+ import
with the second one. In the long run, the osmotic pressure
in the cytoplasm is controlled by compatible solutes,
organic compounds such as the sugar trehalose, which
are synthesized or degraded, imported or exported as a
long-term answer to changing osmotic conditions in the
environment.
Sodium is needed to form its own ion motive force
in addition to or in substitution of the pmf (see Section
S1.2.8). Moreover, it is used to control the internal pH
value, which is usually in mesophilic bacteria close to
pH = 7.6. If the internal pH value changes, a sodium-
proton antiporter substitutes some of the pmf by a
sodium motive force. In the long run, however, the mean
isoelectric point of the cytoplasmic proteome serves a
buer. Should that not be sucient, decarboxylation of
acidic amino acids such as glutamate may keep the pH
value up.
Magnesium is imported by a variety of secondary and
primary uptake systems again, members of the CorA/MIT
(cobalt resistance/metal inorganic transport), MgtE (mag-
nesium transport E) or P-type ATPase protein families,
respectively. If the cytoplasmic magnesium concentration
is too high, magnesium eux may also happen. Calcium
is usually removed from the cytoplasm of bacteria by a
calcium-exporting P-type ATPase.
The metabolism of the minor bioelements or trace ele-
ments is described in Chapter 12.
S1.3.9
Building Blocks
S1.3.9.1 Overview
Biosynthesis of some of the building blocks for the cellular
macromolecules has already been described. The glycerol-
3-phosphate for the synthesis of phospholipids stems from
3GAP (Section S1.3.3.6) and the required fatty acids are
provided as acyl-S-CoA (Section S1.3.6.6). Carbohydrates
including the D-ribose for nucleic acids are synthesized in
the F1,6bP and PPC pathways. This leaves the amino acids
and purine/pyrimidine bases as constituents of the proteins
and nucleic acids. Amino acids are synthesized from F1,6bP
and TCA cycle intermediates in the form of synthesis
families (Figure S1.22). Not all amino acids are used by the

Metal chelator
His Metallothioneins
Ribose-5- Alkaloids
2-Phospho-
glycolate phosphate
(Photorespiration)

Glutathione
Phytochelatins
Gly Ribulose-1,5- Calvin Erythrose-4- Flavonoids
bisphosphate cycle phosphate Lignins
Isophytochelatins Ser Salicylic acid
(SO42 assimilation)
Glutathione 3,5-Bisphospho- Shikimate
glycerate Alkaloids
Phytochelatins Cys
Metallothioneins Phe
Phosphoenolpyruvate Auxines
Antifreeze proteins Ala Chorismate Tyr Indol alkaloids
Brassinosteroide Leu Pyruvate Trp Glucosinolates
receptor Phytoalexins
Acetyl~CoA
Jasmonyl valine Val
Citrate
Pro Osmolyte
N-translocator Asn Gln N-translocator
Oxalo TCA -Keto- Glutathione
Asp Glu
acetate cycle glutarate Phytochelatins
Antifreeze proteins Thr
Jasmonyl isoleucine Ile Polyamines
Orn
Alkaloids Lys Alkaloids
S-Adenosylmethionine Met Polyamines
Arg NO synthesis
Alkaloids

Figure S1.22 Synthesis of amino acids and their derivatives in plants (Graphics: G-J. Krauss, D. Dobritzsch.)

60
W
F
Y
ATP/aa
40 M R
K L
I
C Q V
P
20 T E
N D S A
0
0 20 40
OEC
Figure S1.23 Relationship between costs of an amino acid and
usage in proteins. The costs of a single amino acid is the number
ATP needed to synthesize it from an intermediary compound plus
assimilation of nitrogen plus the amount of ATP that has not been
cell to the same extent; the higher the biosynthesis costs
are, the lower the frequency of occurrence of an amino acid
in a cellular protein (Figure S1.23). If not stated otherwise,
the L-conformation of all amino acids is described.
S1.3.9.2 E, D, Q, N, Acidic Amino Acids and Their Amides
Of the two amino acids that contain a negatively charged
side chain at neutral pH values, glutamate is the primary
product of nitrogen assimilation and source for the amino
groups of most other amino acids (see Section S1.3.8.2).
Aspartate may be synthesized from the TCA cycle com-
pound oxaloacetate in a similar manner as glutamate
by an aspartate dehydrogenase if sucient ammoniac is
present in the cell, or by a transaminase reaction with
glutamate as nitrogen donor. The two amide derivatives
of glutamate and aspartate, glutamine and asparagine,
respectively, are created by ATP-dependent glutamine
or asparagine synthetases in a similar manner (see again
Section S1.3.8.2). So, all four amino acids E, D, Q, and N
stem from the TCA cycle.
S1.3.9.3 Sulfur-Containing Amino Acids C, M
As mentioned in Section S1.3.8.3, H2 S-assimilation of
O-acetyl-serine leads to cysteine. Creation of the second
sulfur-containing amino acid, methionine, is more com-
plicated. It starts with the activation of the C4 carboxylic
group of aspartate by phosphate-transfer from ATP and
subsequent reduction rst to aspartate-4-semialdehyde and
then to homoserine that carries an alcohol group at the
C4 atom. This alcohol group is activated by the transfer
of a succinyl moiety from the TCA cycle intermediate
succinyl-S-CoA to O-succinyl-homoserine, the succinyl
S1.3 Basic Biochemistry S49
G
60 80 100
conserved by the degradation of the intermediate. Here, for E. coli
a yield of 24 ATP per mol of glucose under aerobic conditions has
been assumed. The OEC is the occurrence of the amino acid in the
E. coli proteome normalized to alanine = 100.
moiety is exchanged against cysteine to cystathione, which
is cleaved into pyruvate, ammonium, and homocysteine.
The dierence between homocysteine and methionine
is the methyl group at the sulfur atom. This comes from
N5-methyl-THF and its regeneration is linked to the glycine
biosynthesis (see below).
Methionine itself is the central methyl donor of the
cellular biochemistry and activated for this function by
the transfer to the 5 carbon of ATP leading toe SAM
(S-adenosyl-methionine), phosphate and pyrophosphate.
The sulfur in SAM binds three carbon atoms, is positively
charged, allowing an ecient transfer of the methyl group to
many receptors. The remaining S-adenosyl-homocysteine
is cleaved into adenosine that needs to be phosphory-
lated three times, and homocysteine, closing the cycle
again.
S1.3.9.4 The Alcohols S and T
Threonine is also a product of homoserine and thus related
to methionine and stemming from aspartate. The homoser-
ine is the rst activated of the C4 hydroxide of homoser-
ine, followed by the attachment of homoserine-phosphate
as a Schi base to pyridoxal phosphate. The Schi bond tau-
tomerizes to a C2 imin, which allows together with the good
exit group phosphate an ecient elimination of phosphate,
leading to a respective conjugated double bond between C3
and C4. The threonine synthase now protonates the C4 car-
bon atom, allowing the addition of OH , a tautomerization
back to the Schi base and release of the nished threo-
nine.
Serine stems from the F1,6bP pathway and not from the
TCA cycle. 3-phosphoglycerate is oxidized by NAD+ at the
S50 S1 Basic Biochemical Roots
C2 position to 2-keto-3-phospho glycerate or 3-phospho-
hydroxy pyruvate and is transaminated to 3-phosphoserine
and dephosphorylated to the nal product.
S1.3.9.5 The Conformation Determinants G and P
One of the central functions of glycine and proline in
proteins is the break of an -helical conformation to allow
some exibility in protein folding. Proline stems from
glutamate, and similar to the synthesis of homoserine from
aspartate, the C5 carboxy group of glutamate is activated
by phosphorylation, followed by reduction to C5 glutamate
semialdehyde. In contrast to the shorter aspartate semialde-
hyde, glutamate semialdehyde is able to form a pyrroline
ring structure, -pyrroline-5-caboxylic acid, by an attack
of the amino group on the C5 carboxyl atom, forming an
internal Schi base. This carbonyl-analogous structure
can be easily reduced by hydride transfer from NADPH,
yielding proline.
Glycine is produced by the removal of the C3 atom of
serine to THF leading to N5-N10-methylene-THF, which
can be oxidized to N5-methyl-THF, the methyl-donor
for biosynthesis of methionine and subsequently of most
methyl compounds. As this reaction is reversible, serine
can also be produced from glycine. Together with methio-
nine, serine, and the cofactor THF, glycine is part of the
C1-compound shunt of cellular biochemistry.
So, of the 10 amino acids discussed so far, S, C, and G stem
from the F1,6bP pathway and the remaining 7 amino acids
from the TCA cycle.
S1.3.9.6 Hydrophobic Nonaromatic Amino Acids A, I, V and L
At sucient cytoplasmic ammoniac concentrations, alanine
can be synthesized by an alanine dehydrogenase directly
from pyruvate, or by a transamination reaction using
glutamate as nitrogen donor.
Valine biosynthesis starts like the butandiol fermentation
(Section S1.3.5) by TPP-depending decarboxylation of pyru-
vate and transfer of the hydroxymethyl-moiety from TPP to
a second pyruvate, yielding 2-aceto-lactate. In the fermen-
tation, the compound is now decarboxylated to acetoin but
in valine biosynthesis, the C3 carbonyl group is reduced to
an alcohol by NAD(P)H-mediated hydride transfer to 2,3-
dihydroxy-isovaleric acid. Elimination of water leads to an
enol form that tautomerizes to 2-keto-isovaleric acid, which
can be transaminated to valine.
Biosynthesis of isoleucine is done by the same reactions
and even the same enzymes but with 2-keto-butyric acid
instead of pyruvate (= 2-keto-propionic acid!) as acceptor
of the hydroxymethyl moiety from TPP. Intermediates are
2-aceto-2-hydroxy butyrate; 2,3-dihydroxy-3methylvalric
acid; 2-keto-3-methyl valerate and nally isoleucine. The
2-keto-butyric acid required as starter substance is pro-
duced by the elimination of the amino group of threonine,
followed by the tautomerization of the resulting enol form.
Leucine biosynthesis starts with an intermediate of valine
biosynthesis, 2-keto-isovaleric acid. Similar to butyric acid
fermentation, the C2 CH-acidic carbon of an acetyl-S-
CoA attacks the C2 carbonyl function of 2-keto-isovaleric
acid, leading to 2-isopropylmalate and HS-CoA. Water
elimination and re-addition shifts the hydroxyl function, or
nominally the isopropyl-group from C2 to C3, and oxidation
by NAD+ , and decarboxylation yields 2-keto-isocapronate,
which can be transaminated to leucine.
So, of these four amino acids, alanine, valine, and leucine
stem from the F1,6bP pathway plus acetyl-S-CoA, isoleucine
from this pathway, and the TCA cycle.
S1.3.9.7 Amino Acids with Long Positively Charged Side
Chains, R and K
Arginine stems from glutamate and its synthesis pro-
ceeds via the C5 semialdehyde again, similar to proline,
however, closure to the pyrroline ring is prevented by an
acetyl-S-CoA-dependent acetylation of the amino group of
glutamate to N-acetyl-glutamate. After the attachment of
this protection group, the C5 carboxy function is activated
by phosphorylation, reduced by NADPH to the semialde-
hyde, and transaminated to 2-N-acetyl-ornithine. The
protecting acetyl group can be removed, hydrolyzed, or
used to activate the next glutamate as substrate, yielding
ornithine, an amino acid not used for translation.
The next reaction steps occur also during urea synthe-
sis in animals. First, carbamoyl-phosphate (NH2 CO
phosphate) is synthesized from CO2 , NH3 , and water,
using the energy of 2 ATP, by carbamoyl phosphate syn-
thetase. The NH2 C=O moiety of carbamoyl-phosphate
is attached to the C5 amino group of ornithine, giving
citrulline and phosphate. In an ATP-dependent reaction,
aspartate is transferred to the C=O carbonyl group of the
carbamoyl group to arginino succinate, which is hydrolyzed
into fumarate and arginine. Thus, arginine stems from
glutamate with the additional use of aspartate and net
assimilation of CO2 and NH3 .
Synthesis of lysine is equally complicated and there
are several pathways leading to this amino acid. In the
diaminopimelate pathway, aspartate semialdehyde is
formed and pyruvate added to a ring structure, which is
reduced and opened again by the transfer of succinate from
succinyl-S-CoA. Transamination and removal of succinate
yields 2,6-diaminopimelate and important building mate-
rial for the cell wall of bacteria in the meso or L,L optical
conformation. Simple decarboxylation of the C7 carboxy
group yields lysine.
An alternative biosynthesis pathway transfers an acetyl
moiety from acetyl-S-CoA to 2-ketoglutarate to form
homocitrate. Again reminiscent of TCA cycle reactions,
an aconitase-like reaction creates homoisocitrate, which
is oxidized by NAD(P)+ to oxaloglutarate and decar-
boxylated to 2-keto-adipate. This compound carries one
C atom more than 2-ketoglutarate. Transamination of
the 2-keto-group yields 2-amino-adipate. The terminal
C6 carboxy group is activated by phosphorylation and
reduced by NADPH to 2-amino-adipate semialdehyde. This

compound is not simply transaminated to lysine. Instead,
glutamate is added to the C6 keto group and the resulting
Schi base reduced by NADPH to an intermediate named
saccharopine. Reoxidation by NADP+ leads to cleavage into
2-ketoglutarate and lysine.
So, both amino acids K and R come from TCA cycle inter-
mediates, mainly glutamate.
S1.3.9.8 Aromatic Amino Acids, Y, F and W
Biosynthesis of the aromatic amino acids starts with the
fusion of PEP with erythrose-4-phosphate, components of
the F1,6bP and the PPC. Ring closure, water elimination,
and reduction leads to shikimate, a cyclohexane ring with
one internal double bond, a carboxy group adjacent to the
double bond, and three hydoxyl groups in meta, and para
position to the carboxy group. Phosphorylation, addition
of a second PEP, and dephosphorylation yields chorismate,
the central intermediate for all three aromatic amino
acids. Compared to shikimate, one meta hydroxy group
is eliminated to a second double bond, the second meta
hydroxy group contains an enolpyruvyl moiety, but the para
hydroxy group remains untouched.
For phenylalanine, the chorismate mutase converts the
chorismate to prephenate, which decarboxylates after elim-
ination of water to phenylpyruvate, which now contains an
aromatic ring structure. Transamination of phenylpyruvate
yields phenylalanine.
For tyrosine, reduction of prephenate by NAD+ and sub-
sequent decarboxylation leads to p-hydroxyphenylpyruvate,
which is transaminated to tyrosine.
Synthesis of the indole ring of tryptophane is more com-
plicated. The enolpyruvyl group of chorismate is cleaved
o by transamination to yield anthranilate, benzoic acid
with an amino group in ortho position. The next step needs
5-phospho-ribose-1-pyrophosphate (PRPP). Anthranilate
is added in an N-glyosidic bond to the C1 atom of PRPP and
pyrophosphate is released. The ribose ring is opened, water
is eliminated, and the intermediate decarboxylates under
indole ring formation to indole-3-glycerolephosphate. In
the nal step of the tryptophan synthesis, the 3GAP moiety
of this compound is exchanged for a serine.
S1.3.9.9 Histidine and Purine Bases
Histidine biosynthesis is by far the most complicated amino
acid biosynthesis pathway. In an outstanding initial reac-
tion, PRPP is combined with ATP leading to an N-glycosidic
bond between the C1 atom of the PRPP ribose with the
N1 ring nitrogen of adenine, and release of the PRPP
pyrophosphate rst, followed by pyrophosphate release
from the former ATP. The former purine ring is hydrolyzed,
isomerized, and the amide group from glutamine added
yielding glutamate, a 5-aminoimidazole-4-carboxyamide-
ribonucleotide AICAR and the desired imidazole-glycerole
phosphate. The atoms of the imidazole ring come from
S1.3 Basic Biochemistry S51
the PRPP-ribose (the C=CH group attached to the glyc-
erol phosphate) and the former adenine (the remaining
NHCH=N moiety).
The rest is easy and concerns only the glycerol phos-
phate side chain. Water is eliminated and the resulting
enol compound tautomerizes to a 2-keto group, which is
transaminated to histidinol phosphate. After the release of
the phosphate from the ester bond, the alcohol function in
C1 is oxidized twice by NAD+ to yield histidinal and nally
histidine.
The AICAR serves also as intermediate for the biosyn-
thesis of purine bases. After synthesis of PRPP from ATP
and ribose-5-phosphate, the amide nitrogen of glutamine
attacks the C1 of ribose in an SN2 -reaction yielding
5-phospho-ribolosamine, a ribose containing a phosphate
residue in ester bond at the C5 carbon and an amino
group in an N-glycosidic bond in -position at C1 . In an
ATP-dependent reaction, a glycine residue is attached in an
amide bond to this amino group, followed by the transfer
of a CH=O moiety from N5,N10 methenyl-THF. In a ATP-
and glutamine-dependent reaction, the carbonyl group that
was the carboxy-group of the former glycine receives an
imino group, and another ATP is hydrolyzed to allow ring
formation to 5-phosphoribosyl-5-aminoimidazole. The
aminoimidazole ring is carboxylated at the C4 carbon atom
and this carboxy group receives an amide function from
aspartate in an ATP-dependent reaction that yields AICAR
and fumarate. To close the purine ring, AICAR receives
a formiate in an amide bond from N10-formyl-THF at
the amino function. Elimination of water after the attack
of the remaining amino group on the fresh formyl leads
to the purine ring of inosine in inosine monophosphate,
IMP.
To create AMP, the carbonyl group of IMP, which
comes from CO2 , is transaminated in an interesting
GTP-depending reaction involving aspartate and releasing
fumarate. For GMP, the water is added to the double bond
between the former amino group of AICAR and the former
formyl moiety, reduced to a second carbonyl in xanthosine-
monophosphate XMP, and transaminated to GMP. Now,
glutamine serves as nitrogen donor in an ATP-dependent
reaction.
So, the ring atoms of purine bases are assembled in a step-
wise manner and come from: (i) ammoniac via glutamate;
(ii) glycine; (iii) N5, N10 methenyl-THF; and (iv) ammoniac
via glutamine in the imidazole ring; and (v) CO2 ; (vi) ammo-
niac via aspartate; and (vii) N10-formyl-THF leading to IMP.
S1.3.9.10 Pyrimidine Bases and Pyrrol Rings
Pyrimidine bases stem from aspartate, activated by the
transfer of carbamoyl phosphate to N-carbamoyl aspartate,
followed by ring closure and NAD+ (and avin)-dependent
oxidation to orotic acid, which subsequently reacts with
PRPP to orotidine monophosphate, OMP. Decarboxylation
of the carboxy group at C6 of the pyrimidine ring leads
S52 S1 Basic Biochemical Roots
to UMP. UTP is aminated to CTP in an ATP-dependent
reaction that uses ammoniac directly.
Porphyrin rings stem from a condensation of succinyl-
S-CoA and glycine to 2-amino-3-keto adipate, which
decarboxylates to 5-amino-levulate. Fusion of two 5-amino-
levulate yields porphobilinogen, and condensation of four
of these protoporphyrin IX, the central intermediate for
further synthesis of hemes, F430, B12, or chlorophylls.
S1.3.10
Macromolecules in Bacteria
S1.3.10.1 Nucleic Acids and Transcription
Biosynthesis of the information-carrying macromolecules
RNA and proteins from these building blocks is deter-
mined by the information stored in the DNA. RNA is a
polymer of nucleotide monophosphates (NMP) with the
phosphate groups linking the 5 and 3 carbon atoms of two
ribose units in ester bonds. The four RNA bases are the
pyrimidines U (uracil) and C (cytosine), and the purine
bases G (guanine) and A (adenine). As for a growing RNA
strand the respective next NMP moiety is added at the
3 carbon, RNA grows in the 5 to 3 direction, and the
RNA sequence is noted in this direction. In general, nucleic
acid biosynthesis proceeds always from the 5 end of the
(desoxy) ribose to the 3 end: the 3 carbon atom is always
the receptor of the -phosphate of the following nucleotide
in the synthesis direction.
The dierence between DNA and RNA is a 2 -desoxy
group at the desoxyribose of the DNA that allows the
formation of a double helix and stabilizes the bond
between the nucleotides because formation of 2 ,3 -
phosphate degradation intermediates are prevented.
Second, DNA contains a methylated form of uridine named
thymine.
The information content in DNA is encoded by the
sequence of the four DNA bases adenine A, guanine G,
cytosine C, and thymine T. Similar to RNA, the DNA
sequence is noted in the 5 3 direction. DNA carries
its own back-up system because the information in one
DNA strand is inversely repeated by the second strand
that runs in the opposite direction. As A pairs only with 2
hydrogen bonds with T, and G with 3 of them with C, this
base pairing rule is the prerequisite for DNA replication or
transcription (see below). Because of the obligate pairing of
G-C and A-T, the number of G in a DNA strand is identical
to that of C, and that of A to T; however, the ratio of G+C
to A+T, the GC content, may vary in organisms between
below 30% to above 70%, leading to high-GC or low-GC a DNA strand between a promoter and a terminator. As
organisms.
Chemical modication of a DNA base may lead to prob-
lems with either process but sophisticated cellular repair
mechanisms are able to undo most of these alterations.
Additional mistakes during replication or transcription are
rare because thymine has a lower frequency of being in the
wrong enol tautomeric form than uracil would be because
the positive inductive eect of the methyl group stabilizes
the correct form. This is one reason for T in the DNA
instead of U. The second is that cytosine tends to deaminate
to U so that a U in the DNA is immediately recognized as a
damaged C and can be repaired.
A gene is a region on the DNA that contains the infor-
mation for biosynthesis of a protein or nontranslated RNA.
For gene expression, the information of the DNA needs to
be transcribed into RNA information, a process mediated
by the RNA polymerase. Relying on the same base mecha-
nism of A-T and G-C pairing, one DNA strand is used as a
template for the assembly of an RNA strand that contains
the same information. Promoter regions on the DNA tell
the RNA polymerase which of the two DNA strands is to
be used as the template, and this determines also the direc-
tion of transcription because biosynthesis can occur only in
the 5 3 direction. A terminator determines when to stop
and, therefore, the 3 end of the resulting RNA.
Transcription can be divided into three subevents. In
transcription initiation, the RNA-polymerase binds of
to the promoter, receives the rst two NTP substrates,
forms the rst ester bond by the release of pyrophosphate,
and escapes from the promoter. During transcription
elongation, the RNA polymerase migrates along the DNA
template strand in the 3 5 direction and extends the
RNA strand in 5 3 direction, thereby transforming the
DNA sequence into the corresponding RNA sequence. Note
that this RNA sequence is inverse to that of the template
DNA-strand, but with the exception of U instead of T, iden-
tical to the DNA strand that is not used for transcription.
Therefore, the information of this RNA-analogous DNA
strand can be used to predict the RNA sequence very easily.
Third, during transcription termination, the transcription
may stop if the RNA polymerase reaches a terminator, a
stem-loop structure that folds in the just transcribed part
of the RNA. Both, RNA polymerase and RNA, separate and
leave the DNA.
In eukaryotic organisms, only one gene is usually tran-
scribed and the information can be interrupted by introns
that have to be removed, the mature RNA is further
processed by adding a 5 cap and a 3 poly-A tail, and is
exported from the nucleus to the cytoplasm for transla-
tion (see Section S1.3.12). In prokaryotic organisms, one
RNA strand may contain the information of many genes
(polycistronic RNA) and transcription is followed imme-
diately by translation in the same cellular compartment.
In prokaryotes, an operon is dened as the segment of
such a segment may be transcribed from several promoters
and terminators may not stop transcription with 100%
eciency (and there may be even regulatory events that
control termination eciency), one gene may be part of
several overlapping operons. This allows computation of
many dierent cellular parameters for a quantitative control
of gene expression.

S1.3.10.2 RNA and Translation
Most RNAs in a bacterial cell are ribosomal RNAs (rRNAs,
about 80%). These are the central backbones of the ribo-
somes, which assemble amino acids into a polypeptide
chain (translation) depending on the information of a
messenger RNA (mRNA, about 10%). The transfer RNAs
(tRNAs) bring the amino acids to the translating ribosome.
Small RNAs (sRNAs) are a minor group of RNA that con-
trol a variety of processes nevertheless: (i) one important
protein-sorting pathway; (ii) stability of some mRNAs; (iii)
accessibility of some gene-specic parts of certain mRNAs
for translation; (iv) ordered translation termination if a
mRNA is broken; and (v) other processes.
All RNAs are able to form stem-loop structures com-
posed of a stem of double-stranded RNA topped by a
single-stranded loop of unpaired bases. Stem-loops have a
low frequency of occurrence in DNA. Such a state would
have a higher energy compared to double stranded DNA
because the energy of the hydrogen bonds of the nonpaired
bases is lacking. In all RNAs, except mRNA, the stem-loops
have important structural functions, and higher-order
folding events lead from here to the nal conformation. In
mRNAs they are also important and determine (i) stability
of the RNA; (ii) termination of transcription; (iii) translation
initiation in some examples; (iv) regulatory events such as
attenuation; (v) cotranslational insertion of selenocysteine
or pyrrolysine; and (vi) other processes.
Promoter and terminator determine which of the two
DNA strands is to be transcribed, where to start, and when
to stop. For translation, similar control elements are needed
on the mRNA, and translation also consists of three steps:
initiation, elongation, and termination. In prokaryotes
that are able to use multicistronic mRNAs, a ribosome-
binding site RBS serves as the start signal. As RNA is
single-stranded, no decision concerning the direction of
translation is needed; it runs always from the 5 end of
the mRNA to the 3 end. The RBS is usually composed of
a AGGAGG sequence followed by an AUG after 4 to 9
nucleotides in between. Some bacteria are able to initiate
translation with more degenerated RBS, for example, only
an AGG, GGA, or GAG. Moreover, high-GC bacteria
may use a GUG instead of an AUG for some genes, and
low-GC bacteria likewise an UUG. In such cases, however,
the AGGAGG needs to be better conserved than just an
AGG or such. In general, the better the RBS resembles
AGGAGG-//-AUG, the higher the translation initiation
eciency. In eukaryotes with their monocistronic mRNAs,
usually the rst AUG at the 5 end of the mRNA is used
for translation initiation. In some cases, certain sequence
motifs at the 5 end prevent this and the next AUG is used
instead. Moreover, there are also internal ribosome entry
sites in some, mostly viral, RNAs in eukaryotes.
The nucleotides following the AUG in the 5 3
direction are arranged in triplets, three nucleotides that
determine as a codon the introduction of one of the 20
amino acids into the growing polypeptide chain during
S1.3 Basic Biochemistry S53
elongation (sense codons). Three codons are stop codons
and translation termination happens here; however, two
additional amino acids, seleno-cysteine and pyrrolysine,
may use one of these stop codons as their sense codon. In
such a case, a specic stem-loop structure must be present
to indicate that a specic stop codon shall fulll this sense
function for these two exceptional amino acids. Otherwise,
of the 64 possible codons, three are stop codons (UAA,
UGA, UAG), one means methionine and/or start (AUG),
and one tryptophan (AGG). Obviously, tryptophan is a
recent amino acid that has pirated a former stop codon,
and selenocysteine and pyrrolysine were on their way to do
the same when the genetic code became frozen during
evolution. The remaining 59 codons encode 18 amino
acids. Thus, the genetic code is degenerated, and more
than one codon may indicate a single amino acid. In such
a case, the nucleotide on position 3 is not important for
the determination of the amino acid but may inuence the
translation rate nevertheless.
Ribosomes that perform translation are huge particles
composed of three dierent rRNAs and several dozen
proteins and assigned to a small and large ribosomal
subunit, respectively. They are located in the cytoplasm,
which allows immediate translation after transcription in
prokaryotes. In these organisms, an RBS on the mRNA
(optimally AGGAGG) pairs with an inverse sequence of
the rRNA located in the small subunit (rRNA with a sed-
imentation coecient of 16 Svedberg units or 16S-rRNA).
Initiation factors mediate this process and the subsequent
docking of the large ribosomal or 50S subunit that contains
a 5S and a 23S rRNA, and combines with the 30S small
subunit into the 70S ribosome. Note that Sverdberg units
are not directly related to molecular masses and are not
additive.
Thus, at translation initiation, interaction of the ribo-
some with the RBS on the mRNA positions the start codon
AUG at the peptidyl- or P-site of the ribosome. Before
the large ribosomal subunit docks to the small subunit, a
starter tRNA that carries a methionine is transferred to the
future P-site. In bacteria, the methionine residue carries a
formiate attached to the nitrogen atom of the amino acid
in an amide bond, N-formyl-methionine. Do note that the
fMet-tRNA always is the starter tRNA, that all bacterial
polypeptides initially contain an fMet at the beginning, the
N-terminus, independent of the actual start codon (AUG,
GUG, UUG) used.
An important function of the tRNAs is an outsourcing
of the coding process. Three codons indicate a certain
amino acid to be inserted subsequently but it would take
too much time to decide at the translating ribosome which
amino acid comes next. A tRNA molecule carries at one
end of its structure, which looks like a hand-held hair dryer,
the three nucleotides of the anticodon that pair in the usual
A-U or G-C manner with the codons in the A-site of the
ribosome. At the other end (where the electric cable of the
hair dryer would come out), the 3 end is a CCA nucleotide
S54 S1 Basic Biochemical Roots
sequence. In the loaded tRNA the amino acid is attached
to the 2 or the 3 carbon of the ribose of the terminal A
in an ester bond. Aminoacyl-tRNA synthetases are the
enzymes that decide which of the 22 amino acids are loaded
to more than 22 tRNAs. They bind ATP and their specic
amino acid, form an intermediary mixed acid anhydrite
bond between the -phosphate of AMP and the amino
acid, thereby releasing pyrophosphate, and nally transfer
the amino acid to the 3 -terminal CCA of a bound tRNA
in an ester bond. The tRNA is recognized by its anticodon
region but also by a variety of other surface motifs on the
hair dryer that tell the synthetase that this is a tRNA
in general plus a tRNA for this specic amino acid more
specically.
After the large subunit has docked, the P-site thus
contains the starter tRNA loaded with a peptide-like
structure, the N-formyl-methionine. The three nucleotides
downstream of the ATG start codon are exactly located
in the acceptor or A-site of the ribosome. Mediated by
elongation factors, another loaded tRNA that carries the
next amino acid docks to the A-site. Catalyzed by an
adenine of the large rRNA in the large ribosomal subunit,
the free electron pair of the nitrogen of the amino acid in
the A-site attacks the carbonyl-carbon of the fMet-tRNA
in the P-site, resulting in an empty tRNA in the P-site
and a dipeptide in the A-site. The next step is the translo-
cation, the movement of the ribosome down the mRNA
sequence exactly 3 nucleotides in the 5 3 direction, so
that the A-site codon with the dipeptidyl-tRNA attached
moves into the P-site and the next triplet codon to the
A-site. As other initiation and elongation events, this
needs an elongation factor and energy in the form of GTP.
The empty tRNA does not leave the ribosome immedi-
ately but moves attached to its codon to a third site, the
tRNA-exit site.
The ribosome is now ready for another cycle of trans-
lation elongation. A third loaded tRNA binds the A-site,
thereby substituting the empty tRNA at the t-RNA exit site.
This is very important because the interaction between
ribosome and tRNA is based on many ionic interactions
and hydrogen bonds, not simply the few hydrogen bonds
of the codon-anticodon paring. Simultaneous exchange of
a loaded tRNA at the A-site for an empty tRNA at the exit
site substitutes all these interaction energies except for the
codonanticodon paring. Otherwise, it would not be possi-
ble to discriminate between the small energetic dierences
of correct versus wrong codon-anticodon binding among
all that energetic noise of the other tRNAribosome
interactions.
So, by moving along the mRNA in the 5 3 direction
following the triplett codon pattern, the amino acids
encoded by these codons are assembled into polypeptide,
starting with an fMet in bacteria. The bond between the
amino acids, the peptide bond, is in fact an amide bond
but has a lower energy compared to other amide bonds
because the electrons of the C=O double bond are also
able to delocalize to the nitrogen atom, which is, therefore,
in a partial double bond with the carbon and not able to
rotate around the bonding axis. Translation stops when
a stop codon reaches the A-site, and again termination
factors and GTP are needed to dissemble the translating
complex. The polypeptide is synthesized during this pro-
cess from the amino- or N-terminus (carrying an fMet
in the initial polypeptide in bacteria) to the carboxy- or
C-terminus.
S1.3.10.3 Protein Sorting
During translation, the growing polypeptide chain travels
through the ribosome until it leaves the polypeptide exit
site. Cytoplasmic proteins start to fold into the mature
conformation at this moment, spontaneously or assisted
by helper proteins, chaperones. In many cases, the fMet
and maybe also some of the following N-terminal amino
acids are removed right after translation because the
resulting new N-terminus may determine the stability
of the protein. Finally, homo- or heteromeric proteins are
assembled from the folded polypeptides.
Noncytoplasmic proteins have signal sequences at the
N-terminus, which are recognized by a ribosome-attached
chaperon, the trigger factor, and/or other chaperones
and signal-recognition proteins. Proteins for the general
secretion pore contain a leader sequence with a size of
about 20 amino acyl residues at the N-terminus. Folding is
prevented by chaperones and they are excreted through the
Sec pore complex through a membrane, in case of bacteria
the cytoplasmic membrane. In eukaryotes such signals
determine transport of most proteins into mitochondria or
chloroplasts. After transport, the leader sequence is usually
removed by a signal protease.
Proteins with a high hydrophobic leader are recognized
by a signal recognition particle, translation is interrupted,
the whole ribosome moves to the membrane, translation is
resumed, and the protein excreted again into the Sec pore
cotranslationally, however, not moved through the pore
complex but released into the membrane by lateral opening
of the Sec complex. That way, membrane-integral proteins
reach their target.
A dierent fate awaits proteins with a leader that is longer
than the usual Sec-specic leader and contains a pair of
arginine residues close to the N-terminus. Proteins in this
twin-arginine transport or TAT pathway are allowed
to fold in the cytoplasm. In many cases, cofactors such
as the nickel-containing active site of a hydrogenase, a
molybdenum cofactor MoCo, or a copper site are inserted,
and the nal product is transported through a specic
TAT export pore through the other side of the membrane,
released to the outside, or attached to the outer surface of
the membrane.
There are additional transport pathways for specic
proteins in bacteria, such as proteins of the outer mem-
brane or proteins pumped into a host cell by pathogenic
bacteria. In all these cases, sequence motifs of the

protein determine its fate. When the proteins are in
place they catalyze the already described reactions to
conserve energy, to synthesize building blocks, to tran-
scribe and translate more proteins from single amino
acids, and to sort some proteins into compartments
other than the cytoplasm. Moreover, phospholipids,
the lipopolysaccharides, and cell wall components
of some bacteria as well as storage compounds are
assembled.
S1.3.10.4 The Cellular Metabolic Network
The metabolon of a cell or cellular compartment is the
sum of all the metabolites, building blocks, anabolic and
catabolic compounds that are present at a specic concen-
tration to a given time. Some compounds of this metabolon
control together with signals from the outside of the cell
gene expression and translation via regulatory proteins or
sRNAs. Similarly, activity of key enzymes is controlled.
An example is biosynthesis of amino acids. The genes for
the components of the synthesis pathway are expressed
only if the concentration of the specic amino acid in the
cytoplasm is too low. This is measured by attenuation. Only
when the concentration of the respective loaded tRNA is
too low compared to other tRNAs and the overall energetic
state of the cell, transcription proceeds. Otherwise, it is
aborted. Additional repressor may close down the atten-
uation process if the concentration of the respective free
amino acid is high enough anyhow, serving as a higher level
control. In addition, the enzyme at the beginning of the
pathway measures the concentration of the nal product
carefully, and sometimes additional signals as well, and
pumps metabolites into the biosynthesis pathway only if
the individual amino acid is lacking.
That way, the metabolon controls the transcriptome,
the sum of all RNAs in the cell, and the proteome, the
sum of all proteins. Subsequently, the proteome changes
the metabolon again, leading to another moment in time.
During this process, NTPs are synthesized, used for
transcription, and in parallel for anabolism (ATP), sugar
metabolism (UTP), translation and information processing
(GTP), and fatty acid biosynthesis (CTP), so that all these
processes depend on each other by equilibrated energy
uxes. In bacteria, transcription is immediately followed
by translation. Should translation slow down because of a
slow supply with loaded tRNAs, a signal from the ribosome
decreases the reaction rate of the RNA polymerase, slowing
down both steps of gene expression. Subsequently, the NTP
concentration increases the ow equilibrium, the electron
transport chain, and catabolism slows down, and the
anabololic pathways have a chance to reach the metabolites
and keep up again with the demands of translation.
All this shows that a living cell is a nely tuned network
of macromolecules, metabolites, and information, and each
thread of this net changes continuously with time.
S1.3 Basic Biochemistry S55
S1.3.11
DNA-Replication and Cell Division in Prokaryots
If a cell is big enough, it is time to divide. This is simply
because the power P of a cell, the ux of energy or amount of
energy conserved per time interval, depends on the surface
area of the cell, and the division work W to be done on the
mass of the cell and that on its volume. Thus, if the radius
of a round cell is r, P is proportional to r2 and W to r3 , and
P/W to r2 /r3 = 1/r. So, because the growth rate depends
on P/W , it depends also on 1/r. In other words, the smaller
a cell is, the faster it can grow (See Box S1.1 in Section S1.1).
There are, of course, lower limits of r that have to do with
the ability to react to changes within the cell and outside by
parallel expression of many genes. A cell is as small as it can
get without loosing the ability to harbor sucient ribosomes
for exible parallel-response to stimuli and/or stress.
Division is usually an exceptional event in the life of a cell.
Although bacteria such as E. coli are able to duplicate every
20 min under optimal conditions, starvation rules the nor-
mal life and allows duplication in a natural ecosystem such
as the gut only every 16 h. To nd out when it is time, the
cell volume is controlled in E. coli. If a volume changes but
a concentration remains constant, the number of molecule
changes with the volume. So, all E. coli has to do is to count
the number of a major regulatory protein named DnaA. This
is done by providing a xed number of DnaA-binding sites
at the DNA of the bacterium. If the cell grows, the number of
DnaA increases; the binding sites are occupied one after the
other, until nally the threshold is reached. Now it is time
and DnaA initiates replication of the chromosomal DNA in
E. coli.
For further DNA replication rst desoxy-NTPs (dATP,
dGTP, dCTP, and dTTP) have to be synthesized. There is
always some need for dNTPs in the cell, for example, for
plasmid replication or repair processes, so the activity of
the respective enzymes need only to be upregulated. In E.
coli, a thioredoxin-dependent ribonucleotide diphosphate
reductase uses reduced thioredoxin as electron donor to
reduce the 2 hydroxyl functions of the ribose of the four
NDPs to dNTPs; however, other enzymes may perform a
comparable function in other bacteria and in E. coli under
certain conditions. dUDP is dephosphorylated to dUMP
and the methyl group is attached to the uridine moiety
from N5,N10-methylene-THF. Finally, the triphosphates
are synthesized.
For chromosome replication in bacteria, a highly com-
plicated replication factory is assembled in the middle of
the cell close to the cytoplasmic membrane. The factory
consists of six of DNA-polymerase III, itself a heteromulti-
meric protein. The chromosomal DNA is replicated from
the initiation site simultaneously in both directions in two
replication forks, and three DNA polymerase III complexes
are required for each fork. As DNA polymerization has to
proceed into the 5 3 direction, only one template strand
can be replicated without interruption, the leading strand,
S56 S1 Basic Biochemical Roots
which is replicated by one of the three DNA polymerase
III complexes per fork. Attached to the DNA polymerase
III trimer is a DNA helicase that unwinds the DNA double
strand and transfers the leading strand into the respective
DNA polymerase III complex.
The other strand, the lagging strand, has to be replicated
in segments called Okazaki fragments, and in a direc-
tion opposite to that of the leading strand. The helicase
transfers this lagging strand to the Okazaki primase, a
DNA-dependent RNA polymerase that creates a short
DNA-RNA heteroduplex strand of 10 base pairs as primer
for the subsequent lagging strand replication. This primer
is transferred to a detachable part of the DNA polymerase
III complex, the beta clamp. This beta clamp is a dimer; the
dimer has a donut-like structure and can open for insertion
of DNA. Once closed, this donut remains on the DNA
until released again. The beta clamp serves as a tool-belt
and allows attachment of the remaining subunits of the
DNA polymerase III and also of repair proteins should the
DNA be damaged.
A part of the DNA-polymerase called clamp-loading com-
plex receives the lagging strand from the Okazaki primase
and loads the beta clamp to the primer area. Now, the other
components of the DNA polymerase can bind and replica-
tion in the other direction starts. As one DNA polymerase
works in one direction and the second in the other, a loop of
nished replicated lagging strand starts to appear and grow
at the lagging strand-handling DNA polymerase III com-
plex. This loop grows like a trombone until the DNA poly-
merase reaches the 5 end of the previously nished Okazaki
fragment. At this point, the polymerase detaches from the
beta clamp and is ready for the next Okazaki fragment.
And what is the function of the third DNA polymerase III
complex? While the second is replicating, the next fragment
is prepared and loaded to the third complex. While the third
is replicating, DNA is loaded into the second fragment again.
As initiation and loading takes time but lagging strand syn-
thesis has to keep up with leading strand synthesis, twice as
many DNA polymerase III complexes are needed to repli-
cate the slower strand.
What happens with the primer, the short DNA-RNA het-
eroduplex? Another DNA polymerase, DNA polymerase
I, accepts the DNARNA strand from the beta clamp,
degrades the RNA part, and nishes replication of the
primer region. The nal step is to close the desoxy-ribose-
phosphate backbone, which is done by the DNA-ligase.
There are three other DNA polymerases in E. coli that
are repair polymerases for damaged DNA. They decrease
in their delity of replication from pol II to IV to V but
decrease their sensitivity to damaged substrates. So, DNA
polymerase II replicates small problems with high delity.
If polymerase II cannot handle the problem, polymerase
IV takes over and solves it but with higher error rate. If the
damage is too strong even for polymerase IV, polymerase V,
which is strongly controlled in activity, takes over. Primary
function for polymerase V is to repair the DNA structure to
a double strand with chemically correct bases; it conserves
the information of the DNA if possible. Thus, the action
of polymerase V changes the information content of DNA
with high probability, leading to mutations.
One of the two replication forks reaches the stop sig-
nal, the terminator, earlier than the other. This fork runs
through the terminator and dissembles. When the second
fork reaches the terminator, it runs into the helicase of the
other fork, stops, and dissembles too. At this point, the
DNA daughter strands have to be unwinded and are actively
transported to the poles of the daughter cells to be. During
DNA replication, a division factory has been assembled
at the middle of the cell. The most important factor of
this factory, FtsZ, forms a ring close to the cytoplasmic
membrane at mid-cell and is able to close this ring in an
iris-like manner. When the DNA has been unwinded and
the last part of the DNA molecule has cleared the FtsZ-iris,
it closes completely and the cytoplasm of the two young
daughter cells are separated.
S1.3.12
Genomes and Evolution
Michael Wink
Heidelberg University, Institute of Pharmacy and Molec-
ular Biotechnology, INF 364, 69120 Heidelberg, Germany
S1.3.12.1 Genomes and Their Organization
The genome is dened as the overall number of genes
in an organism. During the past 20 years, quite a
number of genomes from Archaea, Bacteria, and
Eukaryota have been sequenced. Thousands of com-
plete genomic DNA sequences have been documented
(see www.ebi.ac.uk/genomes), among them more than 150
genomes from eukaryotes. The development of new Next
Generation DNA Sequencers during the past 10 years has
revolutionized genomics because these high-throughput
instruments allow a genome analysis in a relatively
short time by now. Therefore, the number of complete
genomes that will become available for evolutionary and
phylogenomic studies is already exceeding 10 000.
Already now, a number of important conclusions can
be drawn, as far as gene numbers and genome size are
concerned. Bacterial genomes contain between 580 000
and 13 million base pairs (bp) (Table S1.7), which encode
between 476 (Mycoplasma genitalium) and 9700 genes
(Sorangium cellulosum). In bacterial genomes most parts
of the DNA represent genes for proteins and RNA (tRNA,
rRNA, small RNAs). Noncoding repetitive DNA is of minor
importance.
In simple eukaryotes, such as yeasts, the genome size
has increased to 14 million bp with over 6300 functional
genes. Filamentous multicellular fungi can reach genome
sizes of 1 billion bp and more than 10 000 genes (Table S1.7,
Figure S1.24). The highly heterogeneous group of protozoa
 S1.3 Basic Biochemistry S57

Table S1.7 Relation between genome size and the number which has led rst to polyploidy and later to a functional
of genes of a few selected species whose genomes have been diploidization. Each duplication has provided an additional
sequenced.
set of genes that could be changed and mutated. In conse-
Organisms Genome sizea) Number of quence, new functions could be generated. As we discuss
base pairs genes later for plants, the multiple copies of genes were probably
Archaea used to develop the genes of secondary metabolism.
Archaeoglobus fulgidus 2.18 106 2405 Genes in prokaryotes have a comparably simple structure
Methanothermobacter 1.75 106 1866 (Figure S1.25). Transcription leads to mRNAs that may
thermoautotrophicus contain the transcripts of one (mono-cistronic mRNA)
Pyrococcus furiosus 1.91 106 2057 or many (poly-cistronic mRNA, also di-, tri-, tetra-,
penta-cistronic, etc.) gene, depending on the individual
Sulfolobus acidocaldarius 2.99 106 2221
operon. In Bacteria, genes for proteins functioning together
Bacteria in one or two interlinked biochemical pathways are often
Clostridium tetani 2.8 106 2373 in an operon, for example, those for the F1 F0 ATPase,
Escherichia coli 4.67 106 4288 those required for the biosynthesis of the amino acid
Hemophilus inuenzae 1.83 106 1702 tryptophan, of antibiotics or polyketides (typical secondary
Mycoplasma genitalium 0.58 106 476 metabolites of certain bacteria, such as Streptomyces
Rhodospirillum rubrum 4.35 106 3791
or Actinomyces of the Actinobacteria). In Archaea the
subunits of heteromultimeric proteins are transcribed
Sorangium cellulosum 13.03 106 9702
together but not so often genes for proteins in the same
Fungi pathway.
Aspergillus fumigatus 2.9 107 9920 Genes in multicellular eukaryotes show a typical
Saccharomyces cerevisiae 1.3 107 6275 intron exon structure (Figure S1.26), in which the
Candida glabrata 1.4 107 5180 exons encode for proteins. Introns are eliminated by
splicing during the mRNA processing. Through alter-
Sporozoa
native splicing (some of the exons are also deleted in
Plasmodium falciparum (causes 2.3 107 5300 dierent tissues) a single gene can produce more than a
malaria)
single protein. Eukaryotic genes are also regulated by a
Plants promoter region to which several transcription factors
Arabidopsis thaliana 1.4 108 26 000 can bind. Only when the correct transcription factors
have bound, RNA polymerase II can start to transcribe
Animals
the corresponding gene (Figure S1.25). Genes encod-
Caenorhabditis elegans (nematode) 1.0 108 20 000
ing enzymes of secondary metabolism in plants are not
Drosophila melanogaster (fruit y) 1.6 108 14 000 organized in gene clusters (as in bacteria) but are indepen-
Danio rerio (zebra sh) 1.0 109 30 000 dently located in dierent regions of the chromosomes.
Mus musculus (mouse) 3.0 109 25 000 This feature makes the search and isolation for such
Homo sapiens (human) 3.2 109 25 000 genes much more complicated and time-consuming in
plants.
a) Haploid genome.

can exceed 100 billion bp, probably because of multiple
genome duplications.
In multicellular animals genome sizes are smaller in inver-
tebrates (insects, nematodes, mollusks) than in vertebrates
(Table S1.7, Figure S1.24). Vertebrates genome encompass
about 13 billion bp and 20 00025 000 genes.
Plant genomes start with 140 million bp (Arabidopsis) and
can be larger than 100 billion bp in some monocots. The
number of functional genes seems to be similar as in ver-
tebrates.
Typical for the genomes of eukaryotes is the presence of a
large amount of repetitive DNA; its function is still largely
unclear. The large genome sizes (Figure S1.23) apparently
evolved through several genome duplications. In plants,
many species were generated through hybridization,
S1.3.12.2 Epigenetics
During the development and dierentiation of multicellular
organisms, part of the genome is permanently silenced
(Figure S1.27). This information is transferred to daughter
cells during mitosis, which express the same degree of
dierentiation. This process is called epigenetic inheritance.
Epigenetic traits are somatic traits that are not inherited to
the next generation.
In eukaryotes, several mechanisms for epigenetic inheri-
tance have been detected. The methylation of the base cyto-
sine to 5-methylcytosine represents a major principle: genes
that are hypermethylated are usually permanently silenced.
Another mechanism concerns the modication of histone
proteins, which together with DNA are organized in nucle-
osomes. Histone proteins carry several amino acid residues
(often of lysine) that can be methylated or acetylated. These
S58 S1 Basic Biochemical Roots

Bacteria / archaea

Fungi

Protozoa

Plants

Insects

Molluscs

Cartilaginous fish

Bony fish

Amphibia

Reptiles

Birds

Mammals

105 106 107 108 109 1010 1011 1012

Base pairs in the haploid genome

Figure S1.24 Genome size variation in dierent groups of organisms.

chromatin modications are also important for gene regu-
lation and epigenetic traits.
It is likely that epigenetic regulation is important in
plantherbivore and plantmicrobe interactions. Details
of the corresponding mechanisms involved in secondary
metabolism have not been explored so far. For many years
plant biotechnologists have tried to produce valuable
secondary metabolites in cell cultures. The cells that lead to
callus and suspension cultures derive from undierentiated
cambium tissue. Although dierentiated tissues usually
produce large amounts of natural products, undierenti-
ated cell cultures often fail to do so. It can be speculated
that the genes for a specic pathway are not activated or
are even inactivated in undierentiated cells, which might
reect epigenetic mechanisms.
S1.3.12.3 Phylogeny Tree of Life
Life began on Planet Earth about 3.5 billion years ago.
The earliest fossils have similarities with Cyanobacteria,
suggesting that early organisms were bacteria that were
able to perform photosynthesis. About 1.62.1 billion years
ago the transition from prokaryotes to eukaryotes took
place, rst by invagination and expansion of the bacterial
cytoplasmic membrane, which formed the nuclear envelope
and internal membrane system (such as ER and Golgi) of
eucytes. The uptake of protobacteria by early eucytes via
endosymbiosis eventually resulted in mitochondria, which
help the eukaryotic cells to produce ATP through the
respiratory chain. When these cells acquired cyanobacteria,
they could perform photosynthesis as well. From early
photosynthetic algal cells, later land plants evolved (see
Chapter 4).
Protobacteria and cyanobacteria imported several thou-
sands of genes to the early eucytes. Most of these genes
(which certainly also included genes for bacterial secondary
metabolism) were transferred to the nucleus by a kind
of horizontal gene transfer (HGT). Mitochondria and
chloroplast still carry circular DNA that encodes a number
of proteins necessary in the respiratory chain (in case of
mitochondria) and photosynthesis (in case of chloroplasts).
In the dierent lineages of algae, there is evidence that
chloroplast endosymbionts independently evolved several
times.
Using nucleotide sequences of marker genes, it was pos-
sible during the past decade to reconstruct the phylogeny of
many animals and plants. These new molecular phylogenies
have a great advantage over earlier phylogenies, which were
constructed using morphological characters, in that conver-
gent traits through adaptive characters no longer obscure
phylogenetic relationships.
 S1.3 Basic Biochemistry S59

Activator protein
Promotor Promotor

TATATT 1 2 3 4 TATA 1

Operator Transcription Enhancer

Transcription start
start
Transcription TFIIF
trp factors
Inactive
RNA-polymerase TFIIA

TFIIH TIFIIB

TATATT
TBP TFIID
RNA polymerase II
Active repressor No transcription
blocks operator

Inactive Mediator complex

repressor
Active
RNA-polymerase
CC
TFIIH
TIFIIB H
TATATT 1 2 3 4 TBP TFIID
mRNA TATA 1
2
mRNA
Protein
1
1 2 3 4 Proteins 1

(a) (b)

Figure S1.25 (a and b) Schematic structure of prokaryotic and eukaryotic genes and gene regulation.

5-upstream Promotor Transcription unit 3-downstream

region region region

Introns

NCS NCS

Enhancer CCAAT- TATA- Enhancer

box box

Exons

Figure S1.26 Structure of a eukaryotic gene with intron and exon structure. NCS = noncoding sequence.
S60 S1 Basic Biochemical Roots

Genetic inheritance Epigenetic inheritance

Gene active Gene active

Mutation Chromatin changes

Gene inactive Gene inactive

Mitosis and cell division

Transmission to somatic cells

Gene inactive Gene inactive Gene inactive Gene inactive

Production of germ cells

Gene inactive Gene active

Figure S1.27 Dierences between genetic and epigenetic inheritance.

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