Bioelectrochemistry 95 (2014) 15–22

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Direct electrochemistry and intramolecular electron transfer of ascorbate oxidase confined 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 Interfacial Nanostructure Research Lab., The Institute of Physical and Chemical Research (RIKEN), 2-1 Hirosawa, Wako-shi, Saitama 351-0198, Japan 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 b c

a r t i c l e

i n f o

Article history: Received 23 April 2013 Received in revised form 7 October 2013 Accepted 8 October 2013 Available online 19 October 2013 Keywords: Ascorbate oxidase Self-assembled monolayer Cysteine Direct electron transfer Intramolecular electron transfer

a b s t r a c t A direct electrochemistry and intramolecular electron transfer of multicopper oxidases are of a great importance for the fabrication of these enzyme-based bioelectrochemical-devices. Ascorbate oxidase from Acremonium sp. (ASOM) has been successfully immobilized via a chemisorptive interaction on the L-cysteine self-assembled monolayer modified gold electrode (cys-SAM/AuE). Thermodynamics and kinetics of adsorption of ASOM on the cys-SAM/AuE were studied using cyclic voltammetry. A well-defined redox wave centered at 166 ± 3 mV (vs. Ag!AgCl!KCl(sat.)) was observed in 5.0 mM phosphate buffer solution (pH 7.0) at the fabricated ASOM electrode, abbreviated as ASOM/cys-SAM/AuE, confirming a direct electrochemistry, i.e., a direct electron transfer (DET) between ASOM and cys-SAM/AuE. The direct electrochemistry of ASOM was further confirmed by taking into account the chemical oxidation of ascorbic acid (AA) by O2 via an intramolecular electron transfer in the ASOM as well as the electrocatalytic oxidation of 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 modified 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 × 10−11 mol cm−2 with the apparent adsorption coefficient of 1.63 × 106 L mol−1. The ASOM confined 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 efficient 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 confined 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; ΓASOM, Surface coverage of ASOM on the electrode surface; ΓsASOM, 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: [email protected] (T. Ohsaka). 1567-5394/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.bioelechem.2013.10.005

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 [2–7]. Thus, the DET of these multicopper oxidases is the key step for the fabrication of these oxidases-based biodevices. The DET of laccase [8–18] and bilirubin oxidase [19–29] has been extensively studied, while there have been only a few papers regarding the DET of ascorbate oxidase [30–33]. Santucci and coworkers reported the DET of ascorbate oxidase at the gold electrode modified with dimeric ascorbate oxidase embedded within a polymeric film of an anionic exchange resin containing tributylmethyl phosphonium chloride (TBMPC) bound to polystyrene, cross-linked with divinylbenzene [31]. It has been also reported that self-assembled monolayer (SAM)-modified gold electrodes are effective for the DET between ascorbate oxidase and gold electrode [30,32]. Sakurai first reported the DET of dimeric ascorbate oxidase using (bis(4-pyridyl) disulphide, bis(2-aminoethyl) disulphide, 3,3′-dithiodipropionic acid and diphenyl disulphide) SAM-modified gold electrodes [30]. Recently,

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B. Patil et al. / Bioelectrochemistry 95 (2014) 15–22

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 (cysSAM) is well known to be effective for the immobilization of enzymes such as superoxide dismutase (SOD) [34–38] and laccase [11] and consequently for realizing their DET. This study is focused on the immobilization of ASOM on the cys-SAM modified 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 [39–45]. 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 confined on the cys-SAM modified gold electrode as well as the intramolecular electron transfer from the T1 site to the T2/T3 site in the ASOM is examined. Thermodynamics and kinetics of ASOM adsorption on the cys-SAM modified gold electrode are also studied.

Milli-Q water (denoted as cys-SAM/AuE). 4 μl of 50 μM 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 temperature and then washed with PBS (5.0 mM, pH 7.0). The thusmodified electrode is referred as ASOM/cys-SAM/AuE. The ASOM/cysSAM/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

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 MΩ·cm) purified by Milli-Q water purification 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 twocompartment 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 otherwise stated. All the measurements were carried out at room temperature (25 ± 1 °C).

Fig. 1 shows the typical cyclic voltammograms (CVs) obtained at bare AuE, cys-SAM/AuE, Apo-ASOM/cys-SAM/AuE and ASOM/cysSAM/AuE in 5.0 mM PBS (pH 7.0) under N2 atmosphere. At a glance, we can see a well-defined 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 ApoASOM/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 reflect 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 (1–100 mV s−1). The Iap and Icp values are proportional to v, but not to v1/2 as expected for the electrode reaction of a surface-confined species (Fig. 2B) [49]. This fact indicates that ASOM is actually confined on the cys-SAM/AuE. According to the Laviron's method[50], from the v dependence of ΔEp (≡Eap − Ecp) at v = 50–400 mV s−1, the electron transfer rate constant (k°) was estimated to be 2.5 ± 2.0 s−1 by assuming that the transfer coefficient (α) is 0.5.

2.2. Cleaning and pretreatment of gold electrode

3.2. Thermodynamics and kinetics of ASOM immobilization

Prior to SAM preparation, AuE was polished first with fine emery paper (#2000, SANKYO, Japan), and then with aqueous slurries of fine alumina powder (1.0 and 0.06 μm) 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 s−1, until the reproducible characteristic cyclic voltammogram (CV) of AuE was obtained.

Fig. 3 shows the adsorption isotherm for ASOM on the cys-SAM/AuE, i.e., the plot of the surface coverage of ASOM (ΓASOM) vs. the bulk concentration of ASOM (CASOM). ΓASOM was calculated using Eq. 1:

2. Experimental 2.1. Materials and instrumentation

2.3. Cysteine self-assembled monolayer (cys-SAM) and ASOM immobilization on cys-SAM modified gold electrode The cys-SAM modified AuE was prepared by immersing the pretreated AuE in 1 mM L-cysteine aqueous solution 1 h at room temperature. The modified electrode was then thoroughly rinsed with

Γ ASOM ¼ Q=nFA

ð1Þ

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) 15–22

c ys-SAM

17

ASOM solution ASOM

T1

1 mM cysteine

T3

Immobilization of ASOM on cys-SAM/AuE

Preparation of cys-SAM /AuE

c ys-SAM/AuE

Bare AuE

T3 T2

ASOM/c ys-SAM/AuE

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

Γ ASOM ¼ Γ

s

ASOM β C ASOM =ð1

þ β C ASOM Þ

ð2Þ

The obtained values of ΓsASOM and β were used in the Frumkin isotherm (Eq. 3) and the best fit was obtained with the interaction parameter, g = 0.5 (Fig. 3, the dashed line). The g value obtained (0.5) suggests a weak attractive interaction between the adsorbed molecules of ASOM. The Gibbs free energy of adsorption, ΔGad, was estimated using Eq. 4 [52]. β C ASOM ¼ fθ=ð1–θÞg expðg θÞ

ð3Þ

ΔGad ¼ −RT ln ð55:6βÞ

ð4Þ

where θ is the fractional coverage of the surface defined as ΓASOM /ΓsASOM and 55.6 is the molar concentration (in mol/L) of water in water (Appendix 1). The value of ΔGad gives a quantitative measure of the adsorption strength of ASOM on the cys-SAM/AuE which was estimated to be −39.7kJmol−1, confirming an interaction between ASOM and the cys-SAM/AuE. Fig. 4A shows the change of ΓASOM as a function of time. Assuming that the adsorption kinetics of ASOM on the cys-SAM/AuE is controlled kinetically (Eq. 5, Appendix 2), the adsorption rate constant, kad, could

be estimated to be 0.9 ± 0.4 L mol−1 s−1 from the plot of ln(1−θ) vs. t which is linear as expected (Fig. 4B). ln ð1−θÞ ¼ −kad C ASOM t

Fig. 5A presents the CVs obtained at the ASOM/cys-SAM/AuE in 5.0 mM PBS of different pH values. The formal potential (E°′) is pHdependent. A plot of the E°′ vs. pH is linear (see Fig. 5B) with a slope of −33 mV/pH which corresponds to a one-proton/two-electron

0.20

A

0.15 0.10 0.05 0.00 -0.05 -0.10 -0.15

-0.20 -0.3 -0.2 -0.1

0.03

T1

2

e

3

-

T3

0.2

0.3

0.4

0.5

4

6

8

10

80

100

I pa

0.02

-

I p / µA

I / µA

0.04

e

2

B

0.03

0.07 1

0.1

(v/ mV s-1)1/2 0.04

0.05

0

E/V (vs. Ag/AgCl) 0

0.06

ð5Þ

3.3. Influence of pH on the direct electron transfer of ASOM

I /µA

preadsorbed and solution solvent molecules, respectively and ASOMad and ASOMsoln represent adsorbed ASOM and ASOM in the solution, respectively. Based on the linearized Langmuir equation (Eq. 2), the saturated surface coverage (ΓsASOM) and the adsorption coefficient, β [51] were determined. Accordingly, a plot of CASOM/ΓASOM vs. CASOM yields 1/ΓsASOM and 1/β ΓsASOM as the slope and intercept, respectively, as shown in Fig. 3B From this plot, ΓsASOM and β were calculated to be 2.41 × 10−11 mol cm−2 and 1.63 × 106 L mol−1, respectively.

T3 T2

4

0.01 0.00 -0.01

0.02 -0.02

0.01

-0.03

0.00 -0.01

0

-0.02 -0.03 -0.3 -0.2 -0.1

I pc

-0.04 20

40

60

v/ mV s-1 0

0.1

0.2

0.3

0.4

0.5

E/V (vs. Ag/AgCl) Fig. 1. CVs obtained at ASOM/cys-SAM/AuE (1), bare AuE (2), cys-SAM/AuE (3) and ApoASOM/cys-SAM/AuE (4) in 5.0 mM PBS (pH 7.0) under N2 atmosphere. v = 10 mV s−1.

Fig. 2. (A) CVs obtained for ASOM/cys-SAM/AuE at different scan rates (1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90 and 100 mV s−1 from inner to outer) in 5.0 mM PBS (pH 7.0) under N2 atmosphere. (B) Plots of anodic and cathodic peak currents (background currentsubtracted, Iap and Icp, respectively) vs. scan rate (solid circles) and square root of scan rate (open circles) (data were taken from Fig. 2A).

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2.5

2.5

A

A

ΓASOM / 10-11 mol cm-2

-11 -2 ASOM/ 10 mol cm

2.0

1.5

1.0

2.0 1.5 1.0 0.5 0.0

0.5

0

4

8

12

16

20

t/h 0.0

0.0 0

20

40

60

80

100

CASOM / µmol L-1

B

-0.5 -1.0

50

-1.5

B

-2.0

40

-2.5 -3.0 -3.5

30

-4.0 -4.5 20

0

5

10

15

20

t/h Fig. 4. (A) Plot of ΓASOM 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 μM ASOM. (B) Plot of ln(1-θ) vs. t.

10

0 0

20

40

60

80

100

CASOM / µmol L-1 Fig. 3. (A) Adsorption isotherms for ASOM on the cys-SAM/AuE at 4 °C and fits 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/ ΓASOM)/105 cm2L−1 = 0.4145 CASOM/μmol L−1 + 2.544 (R2 = 09993).

SAM/AuE. On the other hand, the AA oxidation at the cys-SAM/AuE takes place as a direct reaction at the AuE surface in which AA can reach the Au substrate through a cys-SAM which is not compact. The formal potential at the ascorbic acid/dehydroascorbic acid redox couple

0.03

A

0.02 0.01

I/µA

process. The observed pH dependence of E°′ might be associated with the protonation of a histidine (His 87), as in plastocyanins [53,54], or a residue closely associated with the active site of ASOM as expected for dimeric ascorbate oxidase [31].

0.00 -0.01 -0.02

3.4. Intramolecular electron transfer and direct electrochemistry of ASOM

pH 5.0

pH 6.0

pH 7.0

pH 8.0

-0.04 -0.3 -0.2 -0.1 0.0 0.1 0.2 0.3 0.4 0.5

E/V (vs. Ag/AgCl) 0.25

E°'/V (vs. Ag/AgCl)

The CVs obtained for the oxidation of AA at the cys-SAM/AuE and the 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 figure, at a 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/ cys-SAM/AuE and (ii) the well-defined anodic peak was observed at 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) 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 cysSAM takes place. The first point seems “strange” from the viewpoint of 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 can be understood by the following consideration: the onset potential 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 on the E°′ (0.166 V, see Fig. 2) of the ASOM immobilized on the cys-

-0.03

B

0.20

0.15

0.10 4

5

6

7

8

9

pH Fig. 5. (A) CVs obtained for ASOM/cys-SAM/AuE in 5.0 mM PBS solutions of different pH values. (B) Plots of formal potential (E°′) vs. pH.

B. Patil et al. / Bioelectrochemistry 95 (2014) 15–22

3

I/µA

2

A

Cys-SAM

1 2 3 4 5

AA

e-

DHA

1 AuE

0

-1 -0.3 -0.2 -0.1

0

0.1 0.2 0.3 0.4 0.5

E/V (vs. Ag/AgCl) 1.5

B

1 2 5 4 3

I/µA

1.0

0.5

0.0

-0.5 -0.3 -0.2 -0.1 0

0.1 0.2 0.3 0.4 0.5

E/V (vs. Ag/AgCl) Fig. 6. CVs obtained at the cys-SAM/AuE (A) and the ASOM/cys-SAM/AuE (B) in the solution of 5.0 mM PBS (pH 7.0) containing 1.0 mM AA after 10 (1), 30 (2) and 60 (3) min of O2 bubbling followed by 10 min (4) and 30 min (5) of N2 bubbling in the same solution immediately after the O2 bubbling. v = 10 mV s−1.

AA

19

is −0.14 V vs. Ag!AgCl!KCl(sat.) at pH 7.0 [55] and thus the onset potential of ca. 0 V, which is more negative than that for the ASOMmediated AA oxidation, can be expected for the direct oxidation of AA at the cys-SAM/AuE. It should be noted here that the AA oxidation at the cys-SAM/AuE is shifted to the more negative direction of potential compared with that at the bare AuE, because at the former electrode the cys-SAM prevents the fouling of the electrode surface due to the adsorption of the oxidation product of AA. 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/ AuE, suggesting that ASOM is immobilized (adsorbed) on the cysSAM/AuE in such a way that the access of AA to the active site (i.e., T1 site) of ASOM for its oxidation is not necessarily easy, compared with the case at the cys-SAM/AuE where AA can relatively freely reach the AuE surface through the cys-SAM. During the experiment (in Fig. 6), O2 gas was bubbled in the PBS (pH 7.0) containing 1.0 mM AA for 10, 30 and 60 min and after each bubbling the CVs were measured. Immediately after that N2 gas was 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 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]. 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 anodic current (Ia) at the ASOM/cys-SAM/AuE (Fig. 6B voltammograms 1, 2 and 3) were found to continuously decrease with an increase in the duration of 10 to 60 min O2 bubbling. Further decrease in the Iap was observed in the CVs obtained at the cys-SAM/AuE (Fig. 6A 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

e-

T1 eT3 T3 T2

AA• DHA

eO2

T1 e-

N2 bubbling

O2

T3 T3

H2 O

H2 O

T2

Under O2 atmosphere

B

A

AA

AA• DHA

e-

eT1

T1 T3 T3 T2

Under N2 atmosphere

C

e-

T3 T3 T2

O2 H2 O

Under O2 atmosphere

D

Scheme 2. Fabrication of direct electrochemistry and intramolecular electron transfer of ASOM confined 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 confined on the cys-SAM/AuE in the presence of AA under oxygen atmosphere. (B) Direct electrochemistry and intramolecular electron transfer of ASOM confined 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.

B. Patil et al. / Bioelectrochemistry 95 (2014) 15–22

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 cysSAM/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 reflect 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 cysSAM/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 significant. 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-modified gold electrode, where the enzyme is oriented with the T2 site proximate to the electrode surface, would influence the rate of electron flow 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 confined on the cysSAM/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 the CVs obtained at the ASOM/cys-SAM/AuE for 1.0 mM AA oxidation with different scan rates (i.e. 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90 and 100 mV s−1 from inner to outer).

carboxylic and amino terminal of cys-SAM modified 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 difficult 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 ApoASOM/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

1.0 0.5

0.5

-0.5 -0.3 -0.2 -0.1 0

0.1 0.2 0.3 0.4 0.5

E/V (vs. Ag/AgCl) 0.04

B

0.00

4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 -0.5 -0.3 -0.2 -0.1 0 0.1 0.2 0.3 0.4 0.5

0.025

I/µA

I/µA

-0.04

0.020 0.015

-0.08

e-

0

-

e

-0.12

a

0.010

T1 T3

T3 T2

-0.005

O2 H2O

E/V (vs. Ag/AgCl)

0.1

0.2

0.3

0.4

E/V (vs. Ag/AgCl)

-0.010 -0.015 -0.020

b

-0.025 -0.1 0.0 0.1 0.2 0.3 0.4

-0.16 -0.3 -0.2 -0.1

0.0 -0.5 -0.3

T2

I/µA

I/µA

1.5

I/µA

2.0

T3

T3

0.0

Confined orientation of superoxide dismutase (SOD) on cys-SAM modified AuE was reported to be due to the electrostatic interaction and hydrogen bonding between amino acid residues of SOD and the 2.5

T1

e-

1.0

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

AA• DHA AA

1.5

I/µA

20

E/V (vs. Ag/AgCl)

0.0

0.1

0.2

0.3

0.4

0.5

E/V (vs. Ag/AgCl) -0.2

-0.1

0

0.1

0.2

0.3

0.4

0.5

E/V (vs. Ag/AgCl) Fig. 7. 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 under N2 atmosphere. v = 10 mV s−1. Inset shows plots for the ASOM/cysSAM/AuE at various scan rates (1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90 and 100 mV s−1 from inner to outer) in 5.0 mM PBS (pH 7.0) containing 1.0 mM AA under N2 atmosphere.

Fig. 8. (A) CVs obtained at the ASOM/cys-SAM/AuE in 5.0 mM PBS (pH 7.0) in the absence (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/ cys-SAM/AuE (solid thick line), cys-SAM/AuE (dashed line) and Apo-ASOM/cys-SAM/AuE (dotted line) under O2 atmosphere. v = 10 mV s−1. Insets show anodic (a) and cathodic (b) CVs at the ASOM/cys-SAM/AuE near the redox potential of ASOM immobilized on the cys-SAM/AuE under N2 (solid thin line) and O2 (solid thick line) atmosphere.

B. Patil et al. / Bioelectrochemistry 95 (2014) 15–22

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 modified 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 mol−1 s−1 and the saturated coverage of 2.41 × 10−11 mol cm−2. A fit 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 mol−1). 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 confirming a DET between the ASOM and the cys-SAM/AuE. The ASOM confined 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 finely 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 financially supported by Grant-in-Aid for Scientific 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. References [1] S. Shleev, J. Tkac, A. Christenson, T. Ruzgas, A.I. Yaropolov, J.W. Whittaker, L. Gorton, Direct electron transfer between copper-containing proteins and electrodes, Biosens. Bioelectron. 20 (2005) 2517–2554. [2] A.P. Cole, D.E. Root, P. Mukherjee, E.I. Solomon, T.D.P. Stack, A trinuclear intermediate in the copper-mediated reduction of O2: four electrons from three coppers, Science 273 (1996) 1848–1850. [3] E.I. Solomon, U.M. Sundaram, T.E. Machonkin, Multicopper oxidases and oxygenases, Chem. Rev. 96 (1996) 2563–2605. [4] K. Kataoka, R. Sugiyama, S. Hirota, M. Inoue, K. Urata, Y. Minagawa, D. Seo, T. Sakurai, Four-electron reduction of dioxygen by a multicopper oxidase CueO and roles of Asp112 and Glu506 located adjacent to the trinuclear copper center, J. Biol. Chem. 284 (2009) 14405–14413. [5] A. Messerschmidt, R. Ladenstein, R. Huber, Refined crystal structure of ascorbate oxidase at 1.9 Å resolution, J. Mol. Biol. 224 (1992) 179–205.

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