RUBISCO CATALYZES THE FIXATION OF BOTH CO2 AND O2

The key to photorespiratory CO2 evolution and glycolate metabolism is the bifunctional nature of Rubisco.
In addition to the carboxylation reaction, Rubisco also catalyzes an oxygenase reaction, hence the
name ribulose-1,5-bisphosphate carboxylase-oxygenase. With the addition of a molecule of oxygen,
RuBP is converted into one molecule of 3-PGA and one molecule of phosphoglycolate. The
phosphoglycolate is subsequently metabolized in a series of reactions in the peroxisome and the
mitochondrion that result in the release of a molecule of CO2 and recovery of the remaining carbon by the
PCR cycle. The C2 glycolate cycle, also known as the photosynthetic carbon oxidation (PCO) cycle,
begins with the oxidation of RuBP to 3-PGA and P-glycolate. The 3-PGA is available for further
metabolism by the PCR cycle, but the P-glycolate is rapidly dephosphorylated to glycolate in the
chloroplast. The glycolate is exported from the chloroplast and diffuses to a peroxisome. Taken up by
the peroxisome, the glycolate is oxidized to glyoxylate and hydrogen peroxide. The peroxide is broken
down by catalase and the glyoxylate undergoes a transamination reaction to form the amino acid glycine.
Glycine is then transferred to a mitochondrion where two molecules of glycine (4 carbons) are converted
to one molecule of serine (3 carbons) plus one CO2. Glycine is thus the immediate source of
photorespired CO2. The serine then leaves the mitochondrion, returning to a peroxisome where the amino
group is given up in a transamination reaction and the product, hydroxypyruvate, is reduced to glycerate.
Finally, glycerate is returned to the chloroplast where it is phosphorylated to 3-PGA.
The release of carbon as CO2 during the conversion of glycine to serine is accompanied by the release of
an equivalent amount of nitrogen in the form of ammonia. During active photorespiration, the rate of
ammonia release may be substantially greater than the rate of nitrogen assimilation. This nitrogen is not
lost, however, as the ammonia is rapidly reassimilated in the chloroplast, using the enzymes of the
glutamate synthase cycle.
The C2 glycolate pathway involves complex interactions between photosynthesis, photorespiration, and
various aspects of nitrogen metabolism in at least three different cellular organelles. Much of the
supporting evidence comes from labeling studies employing either 14CO2 or specific intermediates, or
18O2, in which the fate of the label is followed through the various suspected chemical transformations. As
with the PCR cycle, all of the enzymes necessary to carry out the C2 glycolate cycle have been
demonstrated. The distribution of intermediates between the three organelles, however, is not
conclusively established. It is largely inferred from the location of the enzymes. All of the subcellular
organelles involved have been isolated and shown to contain the appropriate enzymes.


WHY PHOTORESPIRATION?
In normal air (21% O2), the rate of photorespiration in sunflower leaves is about 17 percent of gross
photosynthesis. Every photorespired CO2, however, requires an input of two molecules of O2 (Figure
8.16). The true rate of oxygenation is therefore about 34 percent and the ratio of carboxylation to
oxygenation is about 3 to 1 (1.00/0.34). This experimental value agrees with similar values calculated for
several species based on the known characteristic of purified Rubisco. The ratio of carboxylation to
oxygenation depends, however, on the relative levels of O2 and CO2 since both gases compete for
binding at the active site on Rubisco. As the concentration of O2 declines, the relative level of
carboxylation increases until, at zero O2, photorespiration is also zero. On the other hand, increases in
the relative level of O2 (or decrease in CO2) shifts the balance in favor of oxygenation. An increase in
temperature will also favor oxygenation, since as the temperature increases the solubility of gases in
water declines, but O2 solubility is less affected than CO2. Thus O2 will inhibit photosynthesis, measured
by net CO2 reduction, in plants that photorespire. The inhibition of photosynthesis by O2 was first
recognized by Otto Warburg in the 1920s, but 50 years were to pass before the bifunctional nature of
Rubisco offered the first satisfactory explanation for this phenomenon.
There is also an energy cost associated with photorespiration and the glycolate pathway. Not only is the
amount of ATP and NAD(P)H expended in the glycolate pathway following oxygenation (5 ATP + 3
NADPH) greater than that expended for the reduction of one CO2 in the PCR cycle (3 ATP + 2 NADPH),
but there is also a net loss of carbon. On the surface, then, photorespiration appears to be a costly and
inefficient process with respect to both energy and carbon acquisition. It is logical to ask, as many have,
why should the plant indulge in such an apparently wasteful process? This question is not easily
answered, although several ideas have been put forward. One has it that the oxygenase function of
Rubisco is inescapable. Rubisco evolved at a time when the atmosphere contained large amounts of CO2
but little oxygen. Under these conditions, an inability to discriminate between the two gases would have
had little significance to the survival of the organism. BothCO2 andO2 react with the enzyme at the same
active site, and oxygenation requires activation by CO2 just as carboxylation does. It is believed that
oxygen began to accumulate in the atmosphere primarily due to photosynthetic activity, but by the time
the atmospheric content of O2 had increased to significant proportions, the bifunctional nature of the
enzyme had been established without recourse. In a sense, C3 plants were the architect of their own
problemgenerating the oxygen that functions as a competitive inhibitor of carbon reduction. By this
view, then, the oxygenase function is an evolutionary hangover that has no useful role. However, this is
an oversimplified view of photorespiration since photorespiratory mutants of Arabidopsis proved to be
lethal under certain growth conditions, indicating the essential nature of the photorespiratory pathway in
C3 plants. Clearly, any inefficiencies resulting from photorespiration in C3 plants are apparently not
severe. There is no evidence that selection pressures have caused evolution of a form of Rubisco with
lower affinity for O2.
While most agree that oxygenation is an unavoidable consequence of evolution, many have argued that
plants have capitalized on this apparent evolutionary deficiency by turning it into a useful, if not essential,
metabolic sequence. The glycolate pathway, for example, undoubtedly serves a scavenger function. For
each two turns of the cycle, two molecules of phosphoglycolate are formed by oxygenation. Of these four
carbon atoms, one is lost as CO2 and three are returned to the chloroplast. The glycolate pathway thus
recovers 75 percent of the carbon that would otherwise be lost as glycolate. The salvage role alone may
be sufficient justification for the complex glycolate cycle. There is also the possibility that some of the
intermediates, serine and glycine, for example, are of use in other biosynthetic pathways, although this
possibility is still subject to some debate. Recently, strong experimental support has been provided for the
thesis that photorespiration could also function as a sort of safety valve in situations that require
dissipation of excess excitation energy. For example, a significant decline in the photosynthetic capacity
of leaves irradiated in the absence of CO2 and O2 has been reported. Injury is prevented, however, if
sufficient O2 is present to permit photorespiration to occur. Apparently the O2 consumed by
photorespiration is sufficient to protect the plant from photooxidative damage by permitting continued
operation of the electron transport system. This could be of considerable ecological value under
conditions of high light and limited CO2 supply, for example, when the stomata are closed due to moisture
stress. Indeed, photorespiratory mutants of Arabidopsis are more sensitive to photoinhibition than their
wildtype counterparts.
A claim made frequently in the literature is that crop productivity might be significantly enhanced by
inhibiting or genetically eliminating photorespiration. As a result, substantial effort has been expended in
the search for chemicals that inhibit the glycolate pathway or selective breeding for low-photorespiratory
strains. Others have surveyed large numbers of species in an effort to find a Rubisco with a significantly
lower affinity for oxygen. All of these efforts have been unsuccessful, presumably because the basic
premise that photorespiration is detrimental to the plant and counterproductive is incorrect. Clearly,
success in increasing photosynthesis and improving productivity lies in other directions. For example, a
mechanism for concentrating CO2 in the photosynthetic cells could be one way to suppress
photorespiratory loss and improve the overall efficiency of carbon assimilation. That is exactly what has
been achieved by C4 and CAM plants and will be discussed further in Chapter 15.

IN ADDITION TO PCR, CHLOROPLASTS EXHIBIT AN OXIDATIVE PENTOSE PHOSPHATE
CYCLE
Although the oxidative pentose phosphate cycle (OPPC) is restricted to the cytosol in animals, this
pathway is present in both the chloroplast and the cytosol in plants. Furthermore, the chloroplastic OPPC
shares several intermediates with the PCR pathway and is closely integrated with it. The first step in the
oxidative pentose phosphate cycle is the oxidation of glucose-6-P (G-6-P) to 6-phosphogluconate (6-P-
gluconate) by the enzyme glucose-6-phosphate dehydrogenase. The glucose-6-phosphate and
fructose-6-phosphate are components of the same stromal hexose phosphate pool that is shared with the
RPPC. This reaction is highly exergonic (_G<0), and thus is not reversible. As a consequence, this
reaction is apparently the rate-determining step for the stromal OPPC. The second reaction in the OPPC
involves the oxidation of 6-phosphogluconate to ribulose-5-phosphate (R-5-P) by the enzyme
gluconate-6-phosphate dehydrogenase with the production of one molecule of NADPH and one CO2
(Figure 8.17, reaction 2). The simultaneous operation of both the PCR pathway and the OPPC in the
stroma would result in the reduction of one molecule of CO2 to carbohydrate at the expense of three ATP
and two NADPH through the PCR pathway. Subsequently, the carbohydrate would be reoxidized to CO2
by the OPPC yielding two NADPH. Thus, if both metabolic pathways operate simultaneously in the
stroma, three ATP would be consumed with no net fixation of CO2. This would represent futile cycling of
CO2 with the net consumption of ATP. This would be terribly wasteful!
How do plants overcome the apparent conundrum created by the presence of both a reductive and an
oxidative pentose phosphate cycle in the same compartment? The potential for the futile cycling of CO2 is
overcome by metabolic regulation, which ensures that the key enzymes of the PCR cycle are active only
in the light and inactive in the dark. In contrast, the key regulatory enzymes of the OPPC are active only
in the dark. Figure 8.13 shows that key regulatory enzymes of the PCR cycle (FBPase, SBPase and Ru-
5-P kinase) are converted by light from their inactive to their active forms by reduced thioredoxin through
the reducing equivalents generated by photosynthetic electron transport. In contrast to stromal FBPase,
SBPase, and Ru-5-P kinase, which are active when their disulfide
bonds are reduced by thioredoxin (SS SH HS), the key regulatory enzyme in the OPPC
(glucose-6-P dehydrogenase; Figure 8.17, reaction 1) is active when its internal disulfide bonds are
oxidized and inactive when they are reduced by thioredoxin. As a consequence, Rubisco, as well as
stromal FBPase, SBPase, and Ru-5-P kinase are in their active states in the light but phosphogluconate
dehydrogenase is in the inactive state, whereas in the dark, phosphogluconate dehydrogenase is in its
active state and the key enzymes of the PRC pathway are inactive. Thus, this exquisite regulation
ensures that photosynthesis results in the net fixation of CO2 and conversion to carbohydrate and
prevents the wasteful consumption of ATP.
The OPPC is thought to be a means to generate NADPH required to drive biosynthetic reactions such as
lipid and fatty acid biosynthesis in plant mesophyll cells. The oxidative pentose phosphate cycle
represents an important source of pentose phosphate, which serves as a precursor for the ribose and
deoxyribose required in the synthesis of nucleic acids. Another intermediate of the oxidative pentose
phosphate pathway with potential significance to plants is the 4-carbon erythrose-4-P, a precursor for the
biosynthesis of aromatic amino acids, lignin, and flavonoids. In addition, the Ru-5-P generated by the
OPPC in the dark can be converted to RuBP in the light to provide the necessary acceptor molecule to
get the RPPC started.