Bioelectrochemistry 95 (2014) 1522

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Bioelectrochemistry
journal homepage: www.elsevier.com/locate/bioelechem

Direct electrochemistry and intramolecular electron transfer of ascorbate

oxidase conned on L-cysteine self-assembled gold electrode
Bhushan Patil a,b, Yoshiki Kobayashi a, Shigenori Fujikawa b,c, Takeyoshi Okajima a,
Lanqun Mao d, Takeo Ohsaka a,
a
Department of Electronic Chemistry, Interdisciplinary Graduate School of Science and Engineering, Tokyo Institute of Technology, 4259-G1-5 Nagatsuta, Midori-ku, Yokohama 226-8502, Japan
b
Interfacial Nanostructure Research Lab., The Institute of Physical and Chemical Research (RIKEN), 2-1 Hirosawa, Wako-shi, Saitama 351-0198, Japan
c
International Institute for Carbon-Neutral Energy Research (WPI-I2CNER), Kyushu University, 744 Moto-oka, Nishi-ku, Fukuoka 819-0395, Japan
d
Beijing National Laboratory for Molecular Science, Institute of Chemistry, Chinese Academy of Science, 2, Zhongguancun North First Street, Beijing 100190, PR China

a r t i c l e i n f o a b s t r a c t

Article history: A direct electrochemistry and intramolecular electron transfer of multicopper oxidases are of a great importance
Received 23 April 2013 for the fabrication of these enzyme-based bioelectrochemical-devices. Ascorbate oxidase from Acremonium sp.
Received in revised form 7 October 2013 (ASOM) has been successfully immobilized via a chemisorptive interaction on the L-cysteine self-assembled
Accepted 8 October 2013
monolayer modied gold electrode (cys-SAM/AuE). Thermodynamics and kinetics of adsorption of ASOM on
Available online 19 October 2013
the cys-SAM/AuE were studied using cyclic voltammetry.
Keywords:
A well-dened redox wave centered at 166 3 mV (vs. Ag!AgCl!KCl(sat.)) was observed in 5.0 mM phosphate
Ascorbate oxidase buffer solution (pH 7.0) at the fabricated ASOM electrode, abbreviated as ASOM/cys-SAM/AuE, conrming a
Self-assembled monolayer direct electrochemistry, i.e., a direct electron transfer (DET) between ASOM and cys-SAM/AuE. The direct
Cysteine electrochemistry of ASOM was further conrmed by taking into account the chemical oxidation of ascorbic
Direct electron transfer acid (AA) by O2 via an intramolecular electron transfer in the ASOM as well as the electrocatalytic oxidation of
Intramolecular electron transfer AA at the ASOM/cys-SAM/AuE.
Thermodynamics and kinetics of the adsorption of ASOM on the cys-SAM/AuE have been elaborated along with
its direct electron transfer at the modied electrodes on the basis of its intramolecular electron transfer and
electrocatalytic activity towards ascorbic acid oxidation and O2 reduction. ASOM saturated surface area was
obtained as 2.41 1011 mol cm2 with the apparent adsorption coefcient of 1.63 106 L mol1. The ASOM
conned on the cys-SAM/AuE possesses its essential enzymatic function.
2013 Elsevier B.V. All rights reserved.

1. Introduction
A direct electrochemistry of enzymes, i.e., a direct electron transfer
(DET) has attracted much attention for the fabrication of biosensors
and biofuel cells. Thus, understanding of enzyme immobilization
process on electrodes along with its thermodynamics and kinetics is of
great interest [1]. An appropriate enzyme orientation on the electrode
surface is necessary for achieving a DET communication for efcient
electrocatalytic reduction/oxidation processes. Therefore it is important
to modify the surface in such a way that an active site of enzyme can be
Abbreviations: AA, Ascorbic acid; AA, Semidehydroascorbate radical; Apo-ASOM,
Apo form of ascorbate oxidase from Acremonium sp.; AuE, Gold electrode; CASOM,
Concentration of ASOM; C surf AA, Local concentration of AA in the vicinity of the ASOM
conned on the cys-SAM/AuE; C bulk AA, Concentration of AA in the bulk solution; T1,
Type 1 copper active site in the ASOM; T2, Type 2 copper active site in the ASOM; T3,
Type 3 copper active site in the ASOM; I a, Anodic current; ASOM, Surface coverage of
ASOM on the electrode surface; sASOM, Saturated surface coverage of ASOM on the
electrode surface; SOD, Superoxide dismutase.
Corresponding author. Tel.: +81 45 924 5404; fax: +81 45 924 5489.
E-mail address: ohsaka@echem.titech.ac.jp (T. Ohsaka).
in close proximity of the electrode surface without altering its essential
enzymatic activity [1].
Multicopper enzymes (e.g., laccase, bilirubin oxidase and ascorbate
oxidase) have the potential of four-electron oxygen reduction to
water by sequential electron uptake from a reducing substrate such as
phenols and ascorbate [27]. Thus, the DET of these multicopper
oxidases is the key step for the fabrication of these oxidases-based
biodevices. The DET of laccase [818] and bilirubin oxidase [1929]
has been extensively studied, while there have been only a few papers
regarding the DET of ascorbate oxidase [3033]. Santucci and co-
workers reported the DET of ascorbate oxidase at the gold electrode
modied with dimeric ascorbate oxidase embedded within a polymeric
lm of an anionic exchange resin containing tributylmethyl phos-
phonium chloride (TBMPC) bound to polystyrene, cross-linked with
divinylbenzene [31]. It has been also reported that self-assembled
monolayer (SAM)-modied gold electrodes are effective for the DET
between ascorbate oxidase and gold electrode [30,32]. Sakurai rst
reported the DET of dimeric ascorbate oxidase using (bis(4-pyridyl)
disulphide, bis(2-aminoethyl) disulphide, 3,3-dithiodipropionic acid
and diphenyl disulphide) SAM-modied gold electrodes [30]. Recently,

1567-5394/$ see front matter 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.bioelechem.2013.10.005
16 B. Patil et al. / Bioelectrochemistry 95 (2014) 1522
Murata et al. reported a DET between monomeric ascorbate oxidase
from Acremonium sp. HI-25 (ASOM) on a gold electrode is facilitated
by a TBMPC membrane and MPA-SAM (3-mercaptopropionic acid
self-assembled monolayer) combined system [32]. Cysteine-SAM (cys-
SAM) is well known to be effective for the immobilization of enzymes
such as superoxide dismutase (SOD) [3438] and laccase [11] and
consequently for realizing their DET. This study is focused on the
immobilization of ASOM on the cys-SAM modied gold electrode.
Catalytic active centers of ascorbate oxidase are two redox active
moieties, that is, the mononuclear type 1 copper (T1) active site
promoting the oxidation of the electron-donating substrate (i.e.
ascorbic acid (AA)) and the trinuclear copper cluster of type 2 (T2)
and type 3 (T3) sites where water is formed by the four-electron
reduction of molecular O2 [3]. An intramolecular electron transfer
from the T1 site to the T2/T3 cluster has been well realized to be
essential in the enzymatic function of ascorbate oxidases [3945].
A general mechanism describing the electron transfer processes
(including DET and an intramolecular electron transfer) in multicopper
oxidases immobilized on electrodes, which are closely associated with
their enzymatic function, has been discussed[17,18,28,46]. In this
study, the direct electrochemistry of ASOM conned on the cys-SAM
modied gold electrode as well as the intramolecular electron transfer
from the T1 site to the T2/T3 site in the ASOM is examined. Thermo-
dynamics and kinetics of ASOM adsorption on the cys-SAM modied
gold electrode are also studied.
2. Experimental
2.1. Materials and instrumentation
Ascorbate oxidase from Acremonium sp. (T-53) was purchased from
ASAHI KASEI PHARMA (Japan), ascorbic acid from Aldrich, L-cysteine
and all other chemicals from WAKO Pure Chemical Industries, Ltd. and
used as received. All the solutions were prepared using deionized
water (18 Mcm) puried by Milli-Q water purication system
(Millipore, Japan).
Cyclic voltammetric measurements were performed in phosphate
buffer solution (PBS, 5.0 mM, pH 7.0) using an electrochemical
analyzer (ALSCHI 760D, CHI instruments) and a conventional two-
compartment three-electrode electrochemical cell, where Pt wire and
an Ag!AgCl!KCl(sat.) electrodes were used as the counter and reference
electrodes, respectively. Gold electrode (AuE, Bioanalytical Systems Inc.
(BAS); 1.6mm in diameter) served as the working electrode. Electrolyte
solutions were deaerated by bubbling N2 gas into the solution for at
least 30 min prior to each electrochemical measurement unless other-
wise stated. All the measurements were carried out at room tem-
perature (25 1 C).
2.2. Cleaning and pretreatment of gold electrode
Prior to SAM preparation, AuE was polished rst with ne emery
paper (#2000, SANKYO, Japan), and then with aqueous slurries of
ne alumina powder (1.0 and 0.06 m) with the help of a polishing
microcloth. To remove the residual alumina particles the polished
electrode was ultrasonicated in Milli-Q water for 10 min. Finally, the
electrode surface was electrochemically pretreated in 0.1 M H2SO4
solution by successive and multiple potential cycling between 0.2
and +1.5 V vs. Ag!AgCl!KCl(sat.) at 500 mV s1, until the reproducible
characteristic cyclic voltammogram (CV) of AuE was obtained.
2.3. Cysteine self-assembled monolayer (cys-SAM) and ASOM
immobilization on cys-SAM modied gold electrode
The cys-SAM modied AuE was prepared by immersing the
pretreated AuE in 1 mM L-cysteine aqueous solution 1 h at room
temperature. The modied electrode was then thoroughly rinsed with
Milli-Q water (denoted as cys-SAM/AuE). 4 l of 50 M ASOM solution
was casted on the cys-SAM/AuE, and it was kept in a sealed bottle at
4 C for 16 h to avoid the drying of ASOM solution on the cys-SAM/
AuE surface. Then the ASOM solution was air-dried at room tem-
perature and then washed with PBS (5.0 mM, pH 7.0). The thus-
modied electrode is referred as ASOM/cys-SAM/AuE. The ASOM/cys-
SAM/AuE was stable for 2 weeks by storing it in a refrigerator.
The apo enzyme of ASOM (Apo-ASOM) was prepared according to
the reference [47] and it was immobilized on the cys-SAM/AuE
(designated as Apo-ASOM/cys-SAM/AuE) by the similar procedure as
used for the ASOM immobilization. A schematic representation of
ASOM immobilization on the cys-SAM/AuE is presented in Scheme 1.
3. Results and discussion
3.1. Direct electrochemistry of ASOM
Fig. 1 shows the typical cyclic voltammograms (CVs) obtained at
bare AuE, cys-SAM/AuE, Apo-ASOM/cys-SAM/AuE and ASOM/cys-
SAM/AuE in 5.0 mM PBS (pH 7.0) under N2 atmosphere. At a glance,
we can see a well-dened redox wave at the ASOM/cys-SAM/AuE
which is centered at 166 3 mV vs. Ag!AgCl!KCl(sat.), estimated
as (Eap + Ecp) / 2, where Eap and Ecp are the anodic and cathodic peak
potentials, respectively. On the other hand, no redox response was
observed at the other electrodes, i.e., bare AuE, cys-SAM/AuE and Apo-
ASOM/cys-SAM/AuE. A comparison of these voltammograms clearly
demonstrates that the redox wave observed for the ASOM/cys-SAM/
AuE is ascribed to the DET of ASOM. The estimated redox potential
(E = 166 3 mV vs. Ag!AgCl!KCl(sat.)) is ca. 30 mV more negative
than the value measured by a potentiometric titration (i.e. 203 mV vs.
Ag!AgCl!KCl(sat.)) [48]. The present E value is ca. 70 mV negative
compared with the case of the galvanostatically immobilized ASOM
[33] and this difference may reect the different surfaces on which
ASOM is adsorbed, i. e., the cys-SAM and bare Au electrode surfaces,
suggesting that the oxidized form of ASOM is stabilized with respect
to the reduced one at the cys-SAM/AuE surface to a larger degree
compared with that at a bare Au electrode surface. Fig. 2A indicates
the potential scan rate (v) dependence of the redox response observed
at the ASOM/cys-SAM/AuE. The ratios of the anodic peak current
to cathodic one (Iap/Icp) are close to unity in the examined range of v
(1100 mV s1). The Iap and Icp values are proportional to v, but not to
v1/2 as expected for the electrode reaction of a surface-conned species
(Fig. 2B) [49]. This fact indicates that ASOM is actually conned on
the cys-SAM/AuE. According to the Laviron's method[50], from the v
dependence of Ep (Eap Ecp) at v = 50400 mV s1, the electron
transfer rate constant (k) was estimated to be 2.5 2.0 s1 by
assuming that the transfer coefcient () is 0.5.
3.2. Thermodynamics and kinetics of ASOM immobilization
Fig. 3 shows the adsorption isotherm for ASOM on the cys-SAM/AuE,
i.e., the plot of the surface coverage of ASOM (ASOM) vs. the bulk
concentration of ASOM (CASOM). ASOM was calculated using Eq. 1:
ASOM Q=nFA 1
where Q is the amount of charge calculated by the integration of the
cathodic (or anodic) peak current for the reduction (or oxidation) of
the adsorbed ASOM (corrected for the background current), n is
the number of electrons involved in the redox reaction (assumed as
n = 1), F is the Faraday constant and A is the geometric electrode area
(0.020 cm2).
In order for ASOM to adsorb to the cys-SAM/AuE, the displacement
of pre-adsorbed solvent (water) molecules (S) from the electrode
surface is necessary. The adsorption equilibrium can then be expressed
as ASOMsoln + n Sad = ASOMad + n Ssoln, where Sad and Ssoln represent
 B. Patil et al. / Bioelectrochemistry 95 (2014) 1522 17

c ys-SAM ASOM solution

ASOM

T1
1 mM cysteine

Preparation of Immobilization of ASOM on T3

T3
cys-SAM /AuE cys-SAM/AuE T2

Bare AuE c ys-SAM/AuE ASOM/c ys-SAM/AuE

Scheme 1. Schematic representation of ASOM/cys-SAM/AuE.

preadsorbed and solution solvent molecules, respectively and ASOMad be estimated to be 0.9 0.4 L mol1 s1 from the plot of ln(1) vs. t
and ASOMsoln represent adsorbed ASOM and ASOM in the solution, which is linear as expected (Fig. 4B).
respectively. Based on the linearized Langmuir equation (Eq. 2), the
saturated surface coverage (sASOM) and the adsorption coefcient, ln 1 kad C ASOM t 5
[51] were determined. Accordingly, a plot of CASOM/ASOM vs. CASOM
yields 1/sASOM and 1/ sASOM as the slope and intercept, respectively,
as shown in Fig. 3B From this plot, sASOM and were calculated to be
2.41 1011 mol cm2 and 1.63 106 L mol1, respectively. 3.3. Inuence of pH on the direct electron transfer of ASOM

s Fig. 5A presents the CVs obtained at the ASOM/cys-SAM/AuE in

ASOM ASOM C ASOM =1 C ASOM 2
5.0 mM PBS of different pH values. The formal potential (E) is pH-
The obtained values of sASOM and were used in the Frumkin dependent. A plot of the E vs. pH is linear (see Fig. 5B) with a slope
isotherm (Eq. 3) and the best t was obtained with the interaction of 33 mV/pH which corresponds to a one-proton/two-electron
parameter, g = 0.5 (Fig. 3, the dashed line). The g value obtained (0.5)
suggests a weak attractive interaction between the adsorbed molecules
0.20
of ASOM. The Gibbs free energy of adsorption, Gad, was estimated
using Eq. 4 [52]. 0.15 A
C ASOM f=1g expg 3 0.10

0.05
Gad RT ln 55:6 4
I /A

0.00
where is the fractional coverage of the surface dened as ASOM /sASOM -0.05
and 55.6 is the molar concentration (in mol/L) of water in water
(Appendix 1). The value of Gad gives a quantitative measure of the -0.10
adsorption strength of ASOM on the cys-SAM/AuE which was estimated -0.15
to be 39.7kJmol1, conrming an interaction between ASOM and the
cys-SAM/AuE. -0.20
-0.3 -0.2 -0.1 0 0.1 0.2 0.3 0.4 0.5
Fig. 4A shows the change of ASOM as a function of time. Assuming
E/V (vs. Ag/AgCl)
that the adsorption kinetics of ASOM on the cys-SAM/AuE is controlled
kinetically (Eq. 5, Appendix 2), the adsorption rate constant, kad, could (v/ mV s-1)1/2
0 2 4 6 8 10

0.04
B I pa
0.07 0.03
0.06 e
- 0.02
1
0.05 T1 0.01
2
I p / A

-
0.04 e T3
3 T3 0.00
0.03 T2
I / A

4 -0.01
0.02
-0.02
0.01
-0.03
0.00 I pc
-0.01 -0.04
0 20 40 60 80 100
-0.02
v/ mV s-1
-0.03
-0.3 -0.2 -0.1 0 0.1 0.2 0.3 0.4 0.5 Fig. 2. (A) CVs obtained for ASOM/cys-SAM/AuE at different scan rates (1, 5, 10, 20, 30, 40,
E/V (vs. Ag/AgCl) 50, 60, 70, 80, 90 and 100 mV s1 from inner to outer) in 5.0 mM PBS (pH 7.0) under N2
atmosphere. (B) Plots of anodic and cathodic peak currents (background current-
Fig. 1. CVs obtained at ASOM/cys-SAM/AuE (1), bare AuE (2), cys-SAM/AuE (3) and Apo- subtracted, Iap and Icp, respectively) vs. scan rate (solid circles) and square root of scan
ASOM/cys-SAM/AuE (4) in 5.0 mM PBS (pH 7.0) under N2 atmosphere. v = 10 mV s1. rate (open circles) (data were taken from Fig. 2A).
18 B. Patil et al. / Bioelectrochemistry 95 (2014) 1522

2.5 2.5
A A

ASOM / 10-11 mol cm-2

2.0
2.0
-2

1.5
ASOM/ 10 mol cm

1.5
1.0
-11

1.0 0.5

0.5 0.0
0 4 8 12 16 20
t/h

0.0 0.0
0 20 40 60 80 100
CASOM / mol L-1
-0.5 B
-1.0
50
-1.5

B -2.0
40 -2.5
-3.0

30 -3.5
-4.0
-4.5
20 0 5 10 15 20
t/h

10 Fig. 4. (A) Plot of ASOM as a function of time for the adsorption of ASOM on the cys-SAM/
AuE in 5.0 mM PBS (pH 7.0) containing 50 M ASOM. (B) Plot of ln(1-) vs. t.

0
SAM/AuE. On the other hand, the AA oxidation at the cys-SAM/AuE
0 20 40 60 80 100
takes place as a direct reaction at the AuE surface in which AA can
CASOM / mol L-1 reach the Au substrate through a cys-SAM which is not compact. The
formal potential at the ascorbic acid/dehydroascorbic acid redox couple
Fig. 3. (A) Adsorption isotherms for ASOM on the cys-SAM/AuE at 4 C and ts to the
Langmuir (solid line) and Frumkin isotherm (dashed line, with g = 0.5). Sold circles
represent the experimental data. (B) shows the linearized Langmuir isotherm. (CASOM/
ASOM)/105 cm2L1 = 0.4145 CASOM/mol L1 + 2.544 (R2 = 09993). 0.03
A
0.02

process. The observed pH dependence of E might be associated with 0.01

the protonation of a histidine (His 87), as in plastocyanins [53,54], or a 0.00
I/A

residue closely associated with the active site of ASOM as expected for
-0.01
dimeric ascorbate oxidase [31].
-0.02
3.4. Intramolecular electron transfer and direct electrochemistry of ASOM pH 5.0 pH 6.0
-0.03
pH 7.0 pH 8.0
The CVs obtained for the oxidation of AA at the cys-SAM/AuE and the -0.04
-0.3 -0.2 -0.1 0.0 0.1 0.2 0.3 0.4 0.5
ASOM/cys-SAM/AuE in 5.0 mM PBS (pH 7.0) containing 1 mM AA under
the O2 and N2 atmosphere are shown in Fig. 6. From this gure, at a E/V (vs. Ag/AgCl)
glance, we can see that (i) the onset potential of AA oxidation at the
cys-SAM/AuE is by ca. 100 mV more negative than that at the ASOM/ 0.25
cys-SAM/AuE and (ii) the well-dened anodic peak was observed at
B
E'/V (vs. Ag/AgCl)

the cys-SAM/AuE, while the similar anodic peak was not obtained at
the ASOM/cys-SAM/AuE under the examined upper potential (0.45 V) 0.20
which was chosen by considering the fact that the cys-SAM is attached
stably on the AuE, i.e., no oxidative rupture of the Au\S bond of cys-
SAM takes place. The rst point seems strange from the viewpoint of 0.15
so-called enzyme-mediated reaction, because the onset potential for
the ASOM-mediated oxidation of AA is more positive than that for the
unmediated, direct oxidation of AA at the cys-SAM/AuE. However, this 0.10
4 5 6 7 8 9
can be understood by the following consideration: the onset potential
pH
of AA oxidation at the ASOM/cys-SAM/AuE is around 0.11 ~ 0.13 V
which is quite reasonable for the ASOM-mediated AA oxidation based Fig. 5. (A) CVs obtained for ASOM/cys-SAM/AuE in 5.0 mM PBS solutions of different pH
on the E (0.166 V, see Fig. 2) of the ASOM immobilized on the cys- values. (B) Plots of formal potential (E) vs. pH.
 B. Patil et al. / Bioelectrochemistry 95 (2014) 1522 19

3 is 0.14 V vs. Ag!AgCl!KCl(sat.) at pH 7.0 [55] and thus the onset

A Cys-SAM
potential of ca. 0 V, which is more negative than that for the ASOM-
1 mediated AA oxidation, can be expected for the direct oxidation of AA
2 AA
e- 2
3
at the cys-SAM/AuE. It should be noted here that the AA oxidation at
I/A DHA 4 the cys-SAM/AuE is shifted to the more negative direction of potential
1 5 compared with that at the bare AuE, because at the former electrode
AuE the cys-SAM prevents the fouling of the electrode surface due to the
adsorption of the oxidation product of AA.
0
The second point demonstrates that the anodic current for the AA
oxidation is smaller at the ASOM/cys-SAM/AuE than at the cys-SAM/
-1 AuE, suggesting that ASOM is immobilized (adsorbed) on the cys-
-0.3 -0.2 -0.1 0 0.1 0.2 0.3 0.4 0.5 SAM/AuE in such a way that the access of AA to the active site (i.e., T1
E/V (vs. Ag/AgCl) site) of ASOM for its oxidation is not necessarily easy, compared with
1.5 the case at the cys-SAM/AuE where AA can relatively freely reach the
B 1
AuE surface through the cys-SAM.
2 During the experiment (in Fig. 6), O2 gas was bubbled in the PBS
1.0
5 (pH 7.0) containing 1.0 mM AA for 10, 30 and 60 min and after each
4 bubbling the CVs were measured. Immediately after that N2 gas was
I/A

0.5 3 bubbled in the same solution for 10 and 30 min and then the CVs
were measured. It is expected that as the duration of O2 bubbling
0.0 increases, the concentration of AA in the solution decreases gradually
due to the chemical oxidation of AA by oxygen (typically AA + 1/2
O2 DHA + H2O) where DHA is the dehydroascorbic acid [43].
-0.5
-0.3 -0.2 -0.1 0 0.1 0.2 0.3 0.4 0.5
In conformity with this view, the Iap values obtained for oxidation of
AA at the cys-SAM/AuE (Fig. 6A voltammograms 1, 2 and 3) and the
E/V (vs. Ag/AgCl)
anodic current (Ia) at the ASOM/cys-SAM/AuE (Fig. 6B voltammograms
Fig. 6. CVs obtained at the cys-SAM/AuE (A) and the ASOM/cys-SAM/AuE (B) in the 1, 2 and 3) were found to continuously decrease with an increase in
solution of 5.0 mM PBS (pH 7.0) containing 1.0 mM AA after 10 (1), 30 (2) and 60 (3) the duration of 10 to 60 min O2 bubbling. Further decrease in the Iap
min of O2 bubbling followed by 10 min (4) and 30 min (5) of N2 bubbling in the same was observed in the CVs obtained at the cys-SAM/AuE (Fig. 6A
solution immediately after the O2 bubbling. v = 10 mV s1.
voltammograms 4 and 5), while the Ia at the ASOM/cys-SAM/AuE
(Fig. 6B voltammograms 4 and 5) was found to be increased (although

AA DHA
AA AA DHA
AA

e- T1
e- T1
e- O2 N2 bubbling e-
T3 O2
T3 T3
H2 O T3
T2 H2 O
T2

Under O2 atmosphere
B
A

AA DHA
AA e-
T1
e- T1
O2
e- T3
T3 T3
T3 H2 O
T2
T2

Under O2 atmosphere
Under N2 atmosphere

C D
Scheme 2. Fabrication of direct electrochemistry and intramolecular electron transfer of ASOM conned on the cys-SAM/AuE (Entry channel for oxygen is not known, and thus for the easy
understanding of electron transfer it is represented as the channel accessible to the T3 site). AA: semidehydroascorbate radical, DHA: dehydroascorbic acid. (A) Direct electrochemistry
and intramolecular electron transfer of ASOM conned on the cys-SAM/AuE in the presence of AA under oxygen atmosphere. (B) Direct electrochemistry and intramolecular electron
transfer of ASOM conned on the cys-SAM/AuE in the presence of AA under nitrogen bubbling after (A). (C) Oxidation of AA and direct electrochemistry at the ASOM/cys-SAM/AuE
under nitrogen atmosphere. (D) Electrochemistry of ASOM/cys-SAM/AuE under oxygen atmosphere.
20 B. Patil et al. / Bioelectrochemistry 95 (2014) 1522

slightly) with an increase in the duration of N2 bubbling from 10 to
30 min. The further decrease in the Iap after N2 bubbling at the cys-
SAM/AuE is expected due to the chemical oxidation of AA (as
mentioned above) by the molecular oxygen dissolved still in the
solution even after the N2 bubbling, whereas a slight increase in the Ia
at the ASOM/cys-SAM/AuE is considered to reect some additional
facts: It is well known that the electron transfer from the T1 site to the
T2/T3 site is faster under aerobic conditions [5,31,44] and thus the
oxidation of AA by oxygen is enhanced by the ASOM, resulting in the
slowdown of electron transfer from the T1 site to the AuE under O2
atmosphere (Scheme 2A), which might be the cause of a larger decrease
in the Ia obtained at the ASOM/cys-SAM/AuE as compared to the cys-
SAM/AuE. In other words, the intramolecular electron transfer from
the T1 site to the T2/T3 site becomes slower under N2 atmosphere and
relatively the DET from the T1 site to the AuE becomes more signicant.
This is considered to be similar to a transistor-like behavior of a laccase
reported by Shleev and Ruzgas [46] who have mentioned that a
potential applied to the laccase-modied gold electrode, where the
enzyme is oriented with the T2 site proximate to the electrode surface,
would inuence the rate of electron ow between the T1 site andT3
copper site during the enzymatic oxidation of the substrate and
reduction of O2. In our case, it is also considered that the local
concentration of AA in the vicinity of the ASOM conned on the cys-
SAM/AuE (C surf AA) might be slightly increased compared with it
under O2 atmosphere due to the mass transfer of AA from the bulk
solution to the electrode surface by the N2 bubbling, because the bulk
concentration of AA (C bulk AA) is assumed to be slightly higher than
C surf AA under O2 atmosphere. Thus, these might be the cause of a
slight increase in the Ia obtained at the ASOM/cys-SAM/AuE after the
N2 bubbling of 10 and 30 min (Scheme 2 B). A semidehydroascorbate
radical (AA) produced by the electrocatalytic oxidation of AA via the
ASOM undergoes a disproportionation reaction to produce DHA and
AA (typically 2 AA DHA + AA) [3].
Shown in Fig. 7 are the typical CVs obtained at the ASOM/cys-SAM/
AuE in the presence of 0.2, 0.4, 0.6, 0.8 and 1.0 mM AA ensuing an
increase in the I a with increasing the concentration of AA. Inset shows
carboxylic and amino terminal of cys-SAM modied AuE [56]. The pKa
values of the \COO and \NH2 groups of cysteine are 1.71 and
10.78, respectively [57], and thus the cysteine immobilized on the AuE
via S\Au bonding can be expected to behave as zwitterions at pH 7.0.
It has been reported that lysine (Lys) residues are exposed at the surface
of ASOM [32], which have side chains of \NH2. Thus, we may speculate
that the cysteine SAM might be interacted with the ASOM by the
electrostatic interaction between the \COO group of cys-SAM and
the positively charged \NH+ 3 of Lys and by the formation of hydrogen
bond between the NH2 group of cys-SAM and \OH group of serine
(Ser) or threonine (Thr) of ASOM. Such interactions result in the DET
of ASOM as mentioned above. However, so far the molecular structure
of the ASOM is not known [40], and therefore it is difcult to precisely
predict the amino acid residues which may take part in the oriented
adsorption of ASOM on the cys-SAM/AuE surface.
The electrocatalytic activity of the ASOM/cys-SAM/AuE towards the
oxidation of AA and oxygen reduction reaction (ORR) was examined
(Fig. 8). Fig. 8A shows the CVs obtained at the ASOM/cys-SAM/AuE in
the absence (dotted line) and presence (solid line) of AA, which clearly
indicates that the oxidation of AA is catalyzed at the ASOM electrode
(Scheme 2C). The direct reduction of O2 molecules at the cys-SAM/
AuE is shown in Fig. 8B (dashed line). A comparison of the CVs obtained
at the ASOM/cys-SAM under N2 and O2 atmosphere and at the Apo-
ASOM/cys-SAM/AuE under O2 atmosphere demonstrates that the
oxygen reduction is surely catalyzed by the ASOM, although slightly
(see Fig. 8 inset a and b). As shown above, by Fig. 6 and Scheme 2A
and B, this mediated oxygen reduction can be considered to take place
via an intramolecular electron transfer from the T1 site to the T2/T3
site (Scheme 2D). The mediated oxygen reduction via a DET between
the T2/T3 site and the cys-SAM/AuE is also possible, but at the present
stage, we have no direct evidence to support or to deny this possibility.
2.0
A AA DHA
1.5 AA

the CVs obtained at the ASOM/cys-SAM/AuE for 1.0 mM AA oxidation e- T1

with different scan rates (i.e. 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90 and 1.0 T3
I/A

T3
100 mV s1 from inner to outer). T2
0.5

3.5. Orientation of ASOM on the cys-SAM/AuE and its electrocatalytic 0.0

activity
-0.5
-0.3 -0.2 -0.1 0 0.1 0.2 0.3 0.4 0.5
Conned orientation of superoxide dismutase (SOD) on cys-SAM
E/V (vs. Ag/AgCl)
modied AuE was reported to be due to the electrostatic interaction
and hydrogen bonding between amino acid residues of SOD and the 0.04
B
2.5 4.0 0.00
3.5
0.025
2.0 3.0
2.5 -0.04 0.020
I/A

2.0
I/A

I/A

0.015
1.5 1.5
e-
a
0.010
1.0 -0.08 0 0.1 0.2 0.3 0.4
I/A

E/V (vs. Ag/AgCl)

0.5 T1 -0.005
1.0 0.0 - O2 -0.010
e T3
I/A

-0.5 T3 -0.015
-0.3 -0.2 -0.1 0 0.1 0.2 0.3 0.4 0.5 -0.12 T2 H2O
-0.020
0.5 b
E/V (vs. Ag/AgCl) -0.025
-0.1 0.0 0.1 0.2 0.3 0.4
E/V (vs. Ag/AgCl)
-0.16
0.0 -0.3 -0.2 -0.1 0.0 0.1 0.2 0.3 0.4 0.5
E/V (vs. Ag/AgCl)
-0.5
-0.3 -0.2 -0.1 0 0.1 0.2 0.3 0.4 0.5 Fig. 8. (A) CVs obtained at the ASOM/cys-SAM/AuE in 5.0 mM PBS (pH 7.0) in the absence
E/V (vs. Ag/AgCl) (dotted line) and presence (solid line) of 1.0 mM AA under N2 atmosphere. (B) CVs obtained
at the ASOM/cys-SAM/AuE (solid thin line) in 5.0 mM PBS (pH 7.0) under N2 and at ASOM/
Fig. 7. CVs obtained at the ASOM/cys-SAM/AuE in the presence of 0.2, 0.4, 0.6, 0.8 and cys-SAM/AuE (solid thick line), cys-SAM/AuE (dashed line) and Apo-ASOM/cys-SAM/AuE
1.0 mM AA under N2 atmosphere. v = 10 mV s1. Inset shows plots for the ASOM/cys- (dotted line) under O2 atmosphere. v = 10 mV s1. Insets show anodic (a) and cathodic
SAM/AuE at various scan rates (1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90 and 100 mV s1 (b) CVs at the ASOM/cys-SAM/AuE near the redox potential of ASOM immobilized on the
from inner to outer) in 5.0 mM PBS (pH 7.0) containing 1.0 mM AA under N2 atmosphere. cys-SAM/AuE under N2 (solid thin line) and O2 (solid thick line) atmosphere.
 B. Patil et al. / Bioelectrochemistry 95 (2014) 1522
It should be mentioned here that the ORR current observed at the
ASOM/cys-SAM/AuE is too small, compared with that observed for a
usual mediated ORR, probably due to a limited number of the ASOM
immobilized on the cys-SAM/AuE as well as the fact that ASOM is not
necessarily suitably adsorbed on the cys-SAM/AuE in such a way that
O2 molecule can reach freely the active sites (i.e., T2/T3 sites) of ASOM
for its reduction. In addition, in order to illustrate more clearly the
mechanism of the electrocatalytic reaction by the ASOM based on the
DET and an intramolecular electron transfer from the T1 site to the T2/
T3 site as in the case of other multicopper oxidases[17,18,28,46], it is
necessary to obtain the formal potentials(E) of both the T1 and T2/
T3 sites as well as to assign the E value obtained in this study to either
of these both sites.
4. Conclusion
The DET of ASOM on L-cysteine-SAM modied gold electrode has
been achieved. The adsorption of ASOM on the cys-SAM/AuE is kinetically
controlled with the rate constant, kad = 0.9 0.4 L mol1 s1 and the
saturated coverage of 2.41 1011 mol cm2. A t of the experimental
adsorption isotherms to theoretical models indicated the presence of a
slight attractive interaction between ASOM molecules and a large free
energy of adsorption (39.7 kJ mol1). The redox response at the
ASOM/cys-SAM/AuE was found to be pH dependent. An intramolecular
electron transfer in the ASOM and its catalytic activity towards the
AA oxidation under oxygen and nitrogen atmosphere were explored
for conrming a DET between the ASOM and the cys-SAM/AuE.
The ASOM conned on the cys-SAM/AuE was found to possess its
essential enzymatic function. The ASOM/cys-SAM/AuE exhibited an
electromediation activity towards both the oxidation of AA and oxygen
reduction.
A further study regarding a more nely controlled immobilization of
ASOM on the electrode surface, which allows O2 and AA to be freely
accessible to and to react with the respective active sites, i.e., T1 and
T2/T3 sites, respectively, would be needed for developing ASOM-based
electrochemical molecular devices in which the essential enzymatic
reaction can be arbitrarily controlled electrochemically.
Acknowledgments
This research was nancially supported by Grant-in-Aid for
Scientic Research (A) (No. 19206079) to T. Ohsaka from the Ministry
of Education, Culture, Sports, Science and Technology, Japan and
Tokyo Institute of Technology Global COE Program for Energy Science.
An International Program Associate fellowship to B. Patil from RIKEN,
Japan is gratefully acknowledged.
Appendixes 1 and 2. Supplementary data
Supplementary data to this article can be found online at http://dx.
doi.org/10.1016/j.bioelechem.2013.10.005.
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