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Direct heterogeneous electron transfer reactions of bilirubin oxidase at a spectrographic graphite electrode
Electrochemistry Communications 6 (2004) 934–939 www.elsevier.com/locate/elecom
Direct heterogeneous electron transfer reactions of bilirubin oxidase at a spectrographic graphite electrode Sergey Shleev a, Asma El Kasmi b, Tautgirdas Ruzgas c, Lo Gorton
a,*
a
b
Department of Analytical Chemistry, Lund University, P.O. Box 124, SE-221 00 Lund, Sweden School of Science and Engineering, Al Akhawayn University, P.O. Box 1847, 53000 Ifrane, Morocco c Biomedical Laboratory Science, Health and Society, Malmo¨ University, SE-20506 Malmo¨, Sweden Received 15 June 2004; received in revised form 12 July 2004; accepted 12 July 2004 Available online 30 July 2004
Abstract Mediatorless (direct) electron transfer between Myrothecium verrucaria bilirubin oxidase and spectroscopic graphite electrode has been demonstrated. The electrochemical activity of the enzyme under aerobic and anaerobic conditions is clearly shown using cyclic voltammetry. It is concluded that the T1 site of the enzyme is the first electron acceptor, both in solution (homogenous case) and when the bilirubin oxidase is adsorbed on the surface of the graphite electrode (heterogeneous case). Ó 2004 Elsevier B.V. All rights reserved. Keywords: Bilirubin oxidase; Redox potential; T1 site; Carbon electrode; Cyclic voltammetry
1. Introduction Studies of DET reactions between proteins and electrodes yield important information on the thermodynamics and kinetics of biological redox processes. The understanding of the heterogeneous reactions facilitates practical applications of biomolecules in biosensors, biofuel cells, bioelectroorganic synthesis, etc. DET reactions with electrodes have been shown for many proteins [1–5]. Among this extensive group of proteins also some of the larger redox enzymes including multicopper oxidases are found [6–8]. The first publication on DET for a large redox protein with enzymatic activity was about a multicopper
Abbreviations: DET, Direct electron transfer; BOD, Bilirubin oxidase; SPGE, Spectroscopic graphite electrode; PME, Permselective membrane electrode * Corresponding author. Tel.: +46-46-222-7582; fax: +46-46-2224544. E-mail address: [email protected] (L. Gorton). 1388-2481/$ - see front matter Ó 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.elecom.2004.07.008
oxidase, viz. laccase from the basidiomycete Polyporus versicolor [9]. The authors showed that in the presence of molecular oxygen, laccase-modified carbon electrodes exhibited DET. However, under anaerobic conditions the laccase-modified graphite electrode exhibited cyclic voltammograms that were indistinguishable from the background voltammograms obtained in the absence of the enzyme [6,9]. Relatively recently, DET between carbon electrodes and laccases from different sources under anaerobic conditions was confirmed with cyclic voltammetry and square wave voltammetry [10,11]. Data on DET between electrodes and other multicopper oxidases, such as ceruloplasmin, tyrosinase and ascorbate oxidase, were also published [6–8]. Direct bioelectrocatalysis of dioxygen reduction with bilirubin oxidase (BOD) another multicopper oxidase was recently reported [12,13], however, to our best knowledge, there is still no published paper on DET for BOD under anaerobic conditions. Several studies on mediated electrochemistry of the enzyme using different kinds of monomeric mediators as well as redox
S. Shleev et al. / Electrochemistry Communications 6 (2004) 934–939
polymers have been published [14–19]. It was shown that the potential at which the bioelectrocatalytic current starts to appear exclusively depends on the formal potential of the mediator compound employed. The magnitude of the bioelectrocatalytic current systematically decreased with a decrease in the potential difference between E0 0 (O2/H2O) and E0 0 of the mediator [14–19]. From our point of view investigations of DET between electrodes and BOD is a very important task due to several reasons. First of all BOD has the ability of producing mediated bioelectrocatalytic currents for the reduction of O2 to water near neutral pH [19]. This is remarkable compared to previously reported mediated bioelectrocatalytic O2 reductions using laccases that occur at an appreciable magnitude only in acidic solutions (less than pH 5.0). Therefore a biofuel cell utilizing BOD for the four electron reduction of O2 can operate at neutral pH [19–21]. Moreover, BOD has also important biochemical properties for biotechnological application, such as high activity, stability, and low extent of glycosylation [22–24]. BOD has accordingly been utilized in the constructions of biosensors [25,26], of a dioxygen biocathode operating near neutral pH in a H2/ O2 biofuel cell [14,27,28], in a photosynthetic bioelectrochemical cell [15], and in glucose/O2 biofuel cells [18,28,29]. BOD (EC 1.10.3.2) is a multi-copper oxidase catalyzing the oxidation of bilirubin to biliverdin with the concomitant reduction of dioxygen to water [30]. The catalytic site of BOD consists of four copper ions per one enzyme molecule, which can be classified into three types, one type 1 (T1), one type 2 (T2) and two type 3 (T3) copper ions [24,30]. It has been shown that the T1 site is the primary center at which electrons are accepted from reducing substrates [23,30]. BOD from Myrothecium verrucaria was used in the present study for the following reasons. First of all, the enzyme from this source is well characterized. For instance, the biochemical and kinetic properties of M. verrucaria BOD were published in detail [22–24], and the protein sequence is available in the GenBank website (No. B48521). The enzyme has a molecular weight of about 64 kDa [22,30]. A redox potential value of 490 mV vs. NHE was determined at pH 5.3 for the T1 site using potentiometric titrations with redox mediators [23]. The commercial accessibility of M. verrucaria BOD was also a determining factor. Spectroscopic graphite electrodes were preferentially used in our experiments since they were previously shown to electrochemically communicate through DET, under anaerobic and aerobic conditions, with other copper-containing oxidases, such as laccase, tyrosinase, and ceruloplasmine [6,31,32]. Moreover, easy methods for the preparation of enzyme-modified electrodes were developed [6,31,32]. Thus, the main objective of this work was to investigate the possibility of achieving DET for M. verrucaria BOD
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under aerobic and anaerobic conditions using spectroscopic graphite electrodes.
2. Experimental 2.1. Chemicals Na2HPO4, KH2PO4, KCl, NaClO4, and Na2S2O3 were obtained from Merck (Darmstadt, Germany); citric acid was from Sigma Chem. Comp. (St. Louis, MO, USA). All chemicals were of analytical grade. The buffers were prepared using water (18 MX) purified with a Milli-Q system (Millipore, Milford, CT, USA). 2.2. Enzymes BOD from M. verrucaria was received from Sigma and was used with no additional purification. The preparation of BOD was stored at 18 °C. The concentration of BOD in a stock solution was determined by absorbance measurement at 600 nm using an e of 4800 M1 cm1 [24]. 2.3. Electrochemical measurements BOD-modified spectrographic graphite electrodes (SPGE) served as working electrodes. The surface of the SPGE (Ringsdorff Werke GmbH, Bonn, Germany, type RW001, 3.05 diameter, 13% porosity) was prepared by polishing on wet fine emery paper (Tufback Durite, P1200). It was then rinsed thoroughly with Millipore water and allowed to dry. A volume of 10 ll of BOD solution (10 mg/ml) was placed on the electrode surface and allowed to adsorb, and after 15 min the electrode was rinsed again with water. For some experiments a permselective membrane electrode (PME) [33] was prepared by trapping an additional volume of 15 ll of BOD between the SPGE surface and a dialysis membrane (MWCO 6000–8000). The solution of the enzyme was first allowed to dry at the surface of the electrode in air, at room temperature, for 30 min. Then the dialysis membrane (pre-soaked in buffer) was pressed onto the electrode and fitted tight to the electrode with a rubber O-ring. The final enzyme concentration between the electrode and the membrane was approximately 30 mg/ml. Cyclic voltammograms were recorded in a three-electrode electrochemical cell (volume 20 ml) using a potentiostat BAS CV-50W Electrochemical Analyzer controlled with a software v. 2.1 BAS CV-50W (Bioanalytical Systems, West Lafayette, IN). In these experiments an HgjHg2Cl2jKClsat (242 mV vs. NHE) reference electrode and a gold auxiliary electrode were used.
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Anaerobic conditions were established using argon (AGA Gas AB, Sundbyberg, Sweden) purified through a solution containing sodium thiosulfate-5-hydrate.
10
5 1
0
In the presence of the enzyme substrate (molecular oxygen) a reduction current was recorded at a BODmodified SPGE as a result of DET between the electrode and the adsorbed enzyme. Cyclic voltammograms of a bare and a BOD-modified SPGE under aerobic conditions at pH 4.0 are shown in Fig. 1. When adsorbed on SPGE, BOD largely decreased the overvoltage needed for the electroreduction of molecular oxygen. As can be seen from the cyclic voltammogram (Fig. 1), the electrocatalytic current at the electrode modified with BOD starts at a potential of about +800 mV with a half wave potential for oxygen electroreduction of about +700 mV vs. NHE (pH 4.0). The current density is calculated using the geometric electrode surface (0.0731 cm2) corrected with a roughness factor of 5 [34]. A similar behavior was recorded with the BOD-modified PME electrode and electroreduction of oxygen is also possible with this electrode. Moreover, in the case of both modified electrodes, BOD–SPGE and BODPME, a significant catalytic response was found in a broad pH range of the contacting solution, i.e., between pH 3.0 and 9.0. For example, cyclic voltammograms of a BOD-modified PME electrode in deoxygenated and in oxygen-saturated solutions at pH 7.4 are shown in Fig. 2. The current density is calculated in the same way as in Fig. 1. It can be seen that the electrocatalytic current 18 1
13 8 3 j (µA/cm2)
-2 -7 -12 -17 -22 -27 -32 -37 -100
2
100
30 0 500 700 E / mV (vs. NHE)
900
1100
Fig. 1. Electroreduction of molecular oxygen on: (1) a naked SPGE; (2) a SPGE modified with adsorbed BOD. 0.1 M citrate-phosphate buffer pH 4.0; scan rate 10 mV/s; start potential 1000 mV; second scan; aerobic condition (0.26 mM dioxygen).
j (µA/cm2)
3. Results and discussion
-5
-10
-15
2
-20 100
300
500 E / mV (vs. NHE)
700
900
Fig. 2. Electroreduction of molecular oxygen using a PME with adsorbed BOD. 0.2 M phosphate buffer pH 7.4; scan rate 10 mV/s; start potential 900 mV; second scan: (1) in the absence of oxygen; (2) 1.2 mM dioxygen.
at the BOD-modified PME electrode starts at a potential of about +700 mV with a half wave potential for oxygen electroreduction at about +600 mV vs. NHE (pH 7.4). The half wave potential for oxygen electroreduction at pH 7.4 was also determined for BOD–SPGE and found to be equal to +605 mV vs. NHE. This same value was obtained when the experiment was carried in an air-saturated solution (0.2 mM dioxygen) or in an O2-saturated solution (1.2 mM dioxygen). It was also calculated that the half wave potential for oxygen electrocatalytic reduction by BOD is shifted (approximately 180 mV more negative) from the equilibrium O2/H2O potential under these conditions. The possibility for DET between BOD and the electrode under anaerobic conditions was investigated using SPGE. Cyclic voltammograms recorded at sweep rates varying from 1 to 1000 mV/s did not reveal any redox transformation of the enzyme in the potential range between 0 and +1000 mV vs. NHE. Changing the pH from 3 to 9 did not lead to the appearance of any clearly traceable faradic currents on the recorded voltammograms. This result is in good agreement with previously published data concerning the electrochemical behaviour of other multicopper oxidases on SPGE, such as tyrosinase, ascorbate oxidase and laccase [6]. It was possible, however, to observe DET between BOD and the electrode under anaerobic conditions using the PME, e.g., in the case of a high concentration of the enzyme. Fig. 3 shows a background corrected cyclic voltammogram obtained using a BOD-modified PME electrode in a 0.2 M phosphate buffer solution at pH 7.4. The voltammogram shows two small cathodic and anodic peaks respectively at 300 and 730 mV vs. NHE. The mid-
S. Shleev et al. / Electrochemistry Communications 6 (2004) 934–939
1.5
937
20
1 0.5 15 j (µA/cm2)
I (µA)
0 -0.5 -1
10
-1.5 -2 -2.5 200
5
300
400
500 600 700 E / mV (vs. NHE)
800
900
Fig. 3. Cyclic voltammetry (second scan, background correct signal) of a PME with adsorbed BOD obtained under anaerobic conditions. 0.2 M phosphate buffer pH 7.4; scan rate 100 mV/s; start potential +200 mV.
point potential can be estimated to 515 mV, which is very close to the redox potential of the T1 site of the enzyme [23]. The shape of the peaks is suggestive of a diffusionless electron transfer of the adsorbed species. The small electrochemical signal from the enzyme under anaerobic conditions probably relates to the relatively high background noise of carbon electrodes (high capacitive current), which hinders observation of the same process using SPGE. It is anticipated that a small amount of molecular oxygen, which is still present in the pure argon and in the porous electrode, could decrease the visible electrochemical activity of the enzyme under anaerobic conditions. It is well known that multicopper oxidases have a high affinity for molecular oxygen (very low effective Michaelis–Menten constants for oxygen reduction) [30,35,36]. Thus, a small electrocatalytic current can appear and decrease the sensitivity of the electrochemical detection. We would also like to point to the fact that a high irreversibility of the electrochemical process as well as a small electrochemical signal on carbon electrodes was previously shown for other ‘‘blue’’ multicopper oxidase, such as laccase and ceruloplasmine [6,10]. For instance, DET has been reported based on small cyclic voltammetric peaks (background corrected signal) observed for the high potential laccase from P. versicolor under anaerobic conditions [10]. Moreover, in the last few years, a few abstracts (see, for example [37]) have been published, in which DET between oxidases and electrodes has been claimed under anaerobic conditions, however, little or no experimental evidence has been published in peer reviewed journals. Fig. 4 summarizes the dependence of the registered electrocatalytic currents on solution pH. Current densi-
2.5
3 .5
4.5
5 .5
6.5
7 .5
8.5
9.5
pH Fig. 4. Oxygen reduction activity versus pH for a SPGE modified with adsorbed BOD. The BOD activity was determined as the oxygen reduction current density of the BOD modified SPGE at a potential of 0 mV vs. NHE electrode (second scan). About 50 mM citratephosphate buffer; ionic strength 0.2 M (NaClO4); scan rate 25 mV/s; 0.26 mM dioxygen.
ties are plotted versus pH for values ranging from pH 3 to 9. The pH dependence experiment is started after stabilization of the BOD–SPGE achieved by waiting for 1 h in order to get a reproducible signal [38]. The small decay of the activity of the enzyme during the experiment is corrected by using the value at pH 7 recorded at regular intervals. It should also be noticed that the current densities for different pH profile experiments depended on the electrode preparation as well as on batches of the enzyme. Two pH-optima for the reduction of O2 reflecting the catalytic activity of the enzyme can be clearly seen. One could suggest that the two maxima on the pH-profile correspond to different isoforms and/or multiforms of the enzyme in the BOD preparation. As mentioned in Section 2, BOD from M. verrucaria was received from Sigma and was used without any additional purification. However, it is well known that BOD has different pHoptima for different substrates [22,23]. For instance, the enzyme from M. verrucaria showed the highest ABTS oxidation activity at pH 4 [23], whereas the pHoptima for other substrates were in the range from 7 to 8 [22,23]. The obvious similarity between the electrocatalytic reduction of oxygen and the homogeneous activity of BOD indicates that the characteristics of oxygen electrocatalysis are strictly determined by the properties of the enzyme and not by the enzyme-graphite interface. It can also be stated that the decrease in the electrocatalytic activity of the BOD-modified electrodes in the high alkaline region is due to conversion of the active enzyme into an inactive form due to complex formation between T2 Cu2+ and OH [6].
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The obtained results show that the half wave potential for catalytic oxygen electroreduction changes with about 30 mV/pH, whereas the E0 0 (O2/H2O) has a 60 mV/pH dependence. The pH dependence of the T1 site of BOD has not been estimated yet. However, similar results have been demonstrated for the electrocatalytic reduction of O2 by laccase. It was found that the half wave potential for the catalytic oxygen electroreduction by laccases from different origins changes with about 15 mV/pH [32]. A similar pH dependence was previously shown by Nakamura [39] for the redox potential of the T1 site of a low redox potential laccase from Rhus vernicifera and approximately estimated by Reinhammar [40] for a high redox potential laccase from P. versicolor. Moreover, the mentioned similarities of the pH dependencies for the heterogeneous electrocatalysis and the homogeneous characteristics (activity vs. pH, formal potential of the T1 site vs. pH) were also found for laccases from different origins [6,11,32]. Thus, all our data point to the fact that the T1 site is the first electron acceptor in DET from a carbon electrode to the enzyme. The conclusion is based on the fact that the observed dependencies of the electrocatalytic currents on pH for adsorbed BOD closely correlate with those obtained with the corresponding dissolved enzymes for some substrates, where the T1 site is determined to be the first electron acceptor [23,30]. An additional fact that supports this conclusion is that the half wave potential of oxygen electroreduction at graphite electrodes modified with BOD is close to the redox potential of the T1 site of the enzyme when compared with the E0 0 (O2/H2O) under the same conditions. Thus, the conclusion that the T1 site is the first electron acceptor in the enzyme when adsorbed on carbon electrodes under aerobic condition is well motivated. To summarize, all our results with SPGE modified with BOD from M. verrucaria support the possibility of DET between the enzyme and the electrode under aerobic and anaerobic conditions. Moreover, the idea that the T1 site is the first electron acceptor from the electrode in the heterogeneous reduction of the enzyme was confirmed. Our results are in a good agreement with previous data concerning DET processes for other ‘‘blue’’ multicopper oxidases, such as laccase, ascorbate oxidase, and ceruloplasmine [6,8,10,11,32].
Acknowledgements The work was financially supported by the Swedish Research Council, the Russian Foundation for Basic Research (Project No. 03-04-48937), and the Russian Foundation Programme ‘‘Biocatalytic Technologies’’ (Contract No. 43.073.1.1.2505).
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Direct heterogeneous electron transfer reactions of bilirubin oxidase at a spectrographic graphite electrode Sergey Shleev a, Asma El Kasmi b, Tautgirdas Ruzgas c, Lo Gorton
a,*
a
b
Department of Analytical Chemistry, Lund University, P.O. Box 124, SE-221 00 Lund, Sweden School of Science and Engineering, Al Akhawayn University, P.O. Box 1847, 53000 Ifrane, Morocco c Biomedical Laboratory Science, Health and Society, Malmo¨ University, SE-20506 Malmo¨, Sweden Received 15 June 2004; received in revised form 12 July 2004; accepted 12 July 2004 Available online 30 July 2004
Abstract Mediatorless (direct) electron transfer between Myrothecium verrucaria bilirubin oxidase and spectroscopic graphite electrode has been demonstrated. The electrochemical activity of the enzyme under aerobic and anaerobic conditions is clearly shown using cyclic voltammetry. It is concluded that the T1 site of the enzyme is the first electron acceptor, both in solution (homogenous case) and when the bilirubin oxidase is adsorbed on the surface of the graphite electrode (heterogeneous case). Ó 2004 Elsevier B.V. All rights reserved. Keywords: Bilirubin oxidase; Redox potential; T1 site; Carbon electrode; Cyclic voltammetry
1. Introduction Studies of DET reactions between proteins and electrodes yield important information on the thermodynamics and kinetics of biological redox processes. The understanding of the heterogeneous reactions facilitates practical applications of biomolecules in biosensors, biofuel cells, bioelectroorganic synthesis, etc. DET reactions with electrodes have been shown for many proteins [1–5]. Among this extensive group of proteins also some of the larger redox enzymes including multicopper oxidases are found [6–8]. The first publication on DET for a large redox protein with enzymatic activity was about a multicopper
Abbreviations: DET, Direct electron transfer; BOD, Bilirubin oxidase; SPGE, Spectroscopic graphite electrode; PME, Permselective membrane electrode * Corresponding author. Tel.: +46-46-222-7582; fax: +46-46-2224544. E-mail address: [email protected] (L. Gorton). 1388-2481/$ - see front matter Ó 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.elecom.2004.07.008
oxidase, viz. laccase from the basidiomycete Polyporus versicolor [9]. The authors showed that in the presence of molecular oxygen, laccase-modified carbon electrodes exhibited DET. However, under anaerobic conditions the laccase-modified graphite electrode exhibited cyclic voltammograms that were indistinguishable from the background voltammograms obtained in the absence of the enzyme [6,9]. Relatively recently, DET between carbon electrodes and laccases from different sources under anaerobic conditions was confirmed with cyclic voltammetry and square wave voltammetry [10,11]. Data on DET between electrodes and other multicopper oxidases, such as ceruloplasmin, tyrosinase and ascorbate oxidase, were also published [6–8]. Direct bioelectrocatalysis of dioxygen reduction with bilirubin oxidase (BOD) another multicopper oxidase was recently reported [12,13], however, to our best knowledge, there is still no published paper on DET for BOD under anaerobic conditions. Several studies on mediated electrochemistry of the enzyme using different kinds of monomeric mediators as well as redox
S. Shleev et al. / Electrochemistry Communications 6 (2004) 934–939
polymers have been published [14–19]. It was shown that the potential at which the bioelectrocatalytic current starts to appear exclusively depends on the formal potential of the mediator compound employed. The magnitude of the bioelectrocatalytic current systematically decreased with a decrease in the potential difference between E0 0 (O2/H2O) and E0 0 of the mediator [14–19]. From our point of view investigations of DET between electrodes and BOD is a very important task due to several reasons. First of all BOD has the ability of producing mediated bioelectrocatalytic currents for the reduction of O2 to water near neutral pH [19]. This is remarkable compared to previously reported mediated bioelectrocatalytic O2 reductions using laccases that occur at an appreciable magnitude only in acidic solutions (less than pH 5.0). Therefore a biofuel cell utilizing BOD for the four electron reduction of O2 can operate at neutral pH [19–21]. Moreover, BOD has also important biochemical properties for biotechnological application, such as high activity, stability, and low extent of glycosylation [22–24]. BOD has accordingly been utilized in the constructions of biosensors [25,26], of a dioxygen biocathode operating near neutral pH in a H2/ O2 biofuel cell [14,27,28], in a photosynthetic bioelectrochemical cell [15], and in glucose/O2 biofuel cells [18,28,29]. BOD (EC 1.10.3.2) is a multi-copper oxidase catalyzing the oxidation of bilirubin to biliverdin with the concomitant reduction of dioxygen to water [30]. The catalytic site of BOD consists of four copper ions per one enzyme molecule, which can be classified into three types, one type 1 (T1), one type 2 (T2) and two type 3 (T3) copper ions [24,30]. It has been shown that the T1 site is the primary center at which electrons are accepted from reducing substrates [23,30]. BOD from Myrothecium verrucaria was used in the present study for the following reasons. First of all, the enzyme from this source is well characterized. For instance, the biochemical and kinetic properties of M. verrucaria BOD were published in detail [22–24], and the protein sequence is available in the GenBank website (No. B48521). The enzyme has a molecular weight of about 64 kDa [22,30]. A redox potential value of 490 mV vs. NHE was determined at pH 5.3 for the T1 site using potentiometric titrations with redox mediators [23]. The commercial accessibility of M. verrucaria BOD was also a determining factor. Spectroscopic graphite electrodes were preferentially used in our experiments since they were previously shown to electrochemically communicate through DET, under anaerobic and aerobic conditions, with other copper-containing oxidases, such as laccase, tyrosinase, and ceruloplasmine [6,31,32]. Moreover, easy methods for the preparation of enzyme-modified electrodes were developed [6,31,32]. Thus, the main objective of this work was to investigate the possibility of achieving DET for M. verrucaria BOD
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under aerobic and anaerobic conditions using spectroscopic graphite electrodes.
2. Experimental 2.1. Chemicals Na2HPO4, KH2PO4, KCl, NaClO4, and Na2S2O3 were obtained from Merck (Darmstadt, Germany); citric acid was from Sigma Chem. Comp. (St. Louis, MO, USA). All chemicals were of analytical grade. The buffers were prepared using water (18 MX) purified with a Milli-Q system (Millipore, Milford, CT, USA). 2.2. Enzymes BOD from M. verrucaria was received from Sigma and was used with no additional purification. The preparation of BOD was stored at 18 °C. The concentration of BOD in a stock solution was determined by absorbance measurement at 600 nm using an e of 4800 M1 cm1 [24]. 2.3. Electrochemical measurements BOD-modified spectrographic graphite electrodes (SPGE) served as working electrodes. The surface of the SPGE (Ringsdorff Werke GmbH, Bonn, Germany, type RW001, 3.05 diameter, 13% porosity) was prepared by polishing on wet fine emery paper (Tufback Durite, P1200). It was then rinsed thoroughly with Millipore water and allowed to dry. A volume of 10 ll of BOD solution (10 mg/ml) was placed on the electrode surface and allowed to adsorb, and after 15 min the electrode was rinsed again with water. For some experiments a permselective membrane electrode (PME) [33] was prepared by trapping an additional volume of 15 ll of BOD between the SPGE surface and a dialysis membrane (MWCO 6000–8000). The solution of the enzyme was first allowed to dry at the surface of the electrode in air, at room temperature, for 30 min. Then the dialysis membrane (pre-soaked in buffer) was pressed onto the electrode and fitted tight to the electrode with a rubber O-ring. The final enzyme concentration between the electrode and the membrane was approximately 30 mg/ml. Cyclic voltammograms were recorded in a three-electrode electrochemical cell (volume 20 ml) using a potentiostat BAS CV-50W Electrochemical Analyzer controlled with a software v. 2.1 BAS CV-50W (Bioanalytical Systems, West Lafayette, IN). In these experiments an HgjHg2Cl2jKClsat (242 mV vs. NHE) reference electrode and a gold auxiliary electrode were used.
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Anaerobic conditions were established using argon (AGA Gas AB, Sundbyberg, Sweden) purified through a solution containing sodium thiosulfate-5-hydrate.
10
5 1
0
In the presence of the enzyme substrate (molecular oxygen) a reduction current was recorded at a BODmodified SPGE as a result of DET between the electrode and the adsorbed enzyme. Cyclic voltammograms of a bare and a BOD-modified SPGE under aerobic conditions at pH 4.0 are shown in Fig. 1. When adsorbed on SPGE, BOD largely decreased the overvoltage needed for the electroreduction of molecular oxygen. As can be seen from the cyclic voltammogram (Fig. 1), the electrocatalytic current at the electrode modified with BOD starts at a potential of about +800 mV with a half wave potential for oxygen electroreduction of about +700 mV vs. NHE (pH 4.0). The current density is calculated using the geometric electrode surface (0.0731 cm2) corrected with a roughness factor of 5 [34]. A similar behavior was recorded with the BOD-modified PME electrode and electroreduction of oxygen is also possible with this electrode. Moreover, in the case of both modified electrodes, BOD–SPGE and BODPME, a significant catalytic response was found in a broad pH range of the contacting solution, i.e., between pH 3.0 and 9.0. For example, cyclic voltammograms of a BOD-modified PME electrode in deoxygenated and in oxygen-saturated solutions at pH 7.4 are shown in Fig. 2. The current density is calculated in the same way as in Fig. 1. It can be seen that the electrocatalytic current 18 1
13 8 3 j (µA/cm2)
-2 -7 -12 -17 -22 -27 -32 -37 -100
2
100
30 0 500 700 E / mV (vs. NHE)
900
1100
Fig. 1. Electroreduction of molecular oxygen on: (1) a naked SPGE; (2) a SPGE modified with adsorbed BOD. 0.1 M citrate-phosphate buffer pH 4.0; scan rate 10 mV/s; start potential 1000 mV; second scan; aerobic condition (0.26 mM dioxygen).
j (µA/cm2)
3. Results and discussion
-5
-10
-15
2
-20 100
300
500 E / mV (vs. NHE)
700
900
Fig. 2. Electroreduction of molecular oxygen using a PME with adsorbed BOD. 0.2 M phosphate buffer pH 7.4; scan rate 10 mV/s; start potential 900 mV; second scan: (1) in the absence of oxygen; (2) 1.2 mM dioxygen.
at the BOD-modified PME electrode starts at a potential of about +700 mV with a half wave potential for oxygen electroreduction at about +600 mV vs. NHE (pH 7.4). The half wave potential for oxygen electroreduction at pH 7.4 was also determined for BOD–SPGE and found to be equal to +605 mV vs. NHE. This same value was obtained when the experiment was carried in an air-saturated solution (0.2 mM dioxygen) or in an O2-saturated solution (1.2 mM dioxygen). It was also calculated that the half wave potential for oxygen electrocatalytic reduction by BOD is shifted (approximately 180 mV more negative) from the equilibrium O2/H2O potential under these conditions. The possibility for DET between BOD and the electrode under anaerobic conditions was investigated using SPGE. Cyclic voltammograms recorded at sweep rates varying from 1 to 1000 mV/s did not reveal any redox transformation of the enzyme in the potential range between 0 and +1000 mV vs. NHE. Changing the pH from 3 to 9 did not lead to the appearance of any clearly traceable faradic currents on the recorded voltammograms. This result is in good agreement with previously published data concerning the electrochemical behaviour of other multicopper oxidases on SPGE, such as tyrosinase, ascorbate oxidase and laccase [6]. It was possible, however, to observe DET between BOD and the electrode under anaerobic conditions using the PME, e.g., in the case of a high concentration of the enzyme. Fig. 3 shows a background corrected cyclic voltammogram obtained using a BOD-modified PME electrode in a 0.2 M phosphate buffer solution at pH 7.4. The voltammogram shows two small cathodic and anodic peaks respectively at 300 and 730 mV vs. NHE. The mid-
S. Shleev et al. / Electrochemistry Communications 6 (2004) 934–939
1.5
937
20
1 0.5 15 j (µA/cm2)
I (µA)
0 -0.5 -1
10
-1.5 -2 -2.5 200
5
300
400
500 600 700 E / mV (vs. NHE)
800
900
Fig. 3. Cyclic voltammetry (second scan, background correct signal) of a PME with adsorbed BOD obtained under anaerobic conditions. 0.2 M phosphate buffer pH 7.4; scan rate 100 mV/s; start potential +200 mV.
point potential can be estimated to 515 mV, which is very close to the redox potential of the T1 site of the enzyme [23]. The shape of the peaks is suggestive of a diffusionless electron transfer of the adsorbed species. The small electrochemical signal from the enzyme under anaerobic conditions probably relates to the relatively high background noise of carbon electrodes (high capacitive current), which hinders observation of the same process using SPGE. It is anticipated that a small amount of molecular oxygen, which is still present in the pure argon and in the porous electrode, could decrease the visible electrochemical activity of the enzyme under anaerobic conditions. It is well known that multicopper oxidases have a high affinity for molecular oxygen (very low effective Michaelis–Menten constants for oxygen reduction) [30,35,36]. Thus, a small electrocatalytic current can appear and decrease the sensitivity of the electrochemical detection. We would also like to point to the fact that a high irreversibility of the electrochemical process as well as a small electrochemical signal on carbon electrodes was previously shown for other ‘‘blue’’ multicopper oxidase, such as laccase and ceruloplasmine [6,10]. For instance, DET has been reported based on small cyclic voltammetric peaks (background corrected signal) observed for the high potential laccase from P. versicolor under anaerobic conditions [10]. Moreover, in the last few years, a few abstracts (see, for example [37]) have been published, in which DET between oxidases and electrodes has been claimed under anaerobic conditions, however, little or no experimental evidence has been published in peer reviewed journals. Fig. 4 summarizes the dependence of the registered electrocatalytic currents on solution pH. Current densi-
2.5
3 .5
4.5
5 .5
6.5
7 .5
8.5
9.5
pH Fig. 4. Oxygen reduction activity versus pH for a SPGE modified with adsorbed BOD. The BOD activity was determined as the oxygen reduction current density of the BOD modified SPGE at a potential of 0 mV vs. NHE electrode (second scan). About 50 mM citratephosphate buffer; ionic strength 0.2 M (NaClO4); scan rate 25 mV/s; 0.26 mM dioxygen.
ties are plotted versus pH for values ranging from pH 3 to 9. The pH dependence experiment is started after stabilization of the BOD–SPGE achieved by waiting for 1 h in order to get a reproducible signal [38]. The small decay of the activity of the enzyme during the experiment is corrected by using the value at pH 7 recorded at regular intervals. It should also be noticed that the current densities for different pH profile experiments depended on the electrode preparation as well as on batches of the enzyme. Two pH-optima for the reduction of O2 reflecting the catalytic activity of the enzyme can be clearly seen. One could suggest that the two maxima on the pH-profile correspond to different isoforms and/or multiforms of the enzyme in the BOD preparation. As mentioned in Section 2, BOD from M. verrucaria was received from Sigma and was used without any additional purification. However, it is well known that BOD has different pHoptima for different substrates [22,23]. For instance, the enzyme from M. verrucaria showed the highest ABTS oxidation activity at pH 4 [23], whereas the pHoptima for other substrates were in the range from 7 to 8 [22,23]. The obvious similarity between the electrocatalytic reduction of oxygen and the homogeneous activity of BOD indicates that the characteristics of oxygen electrocatalysis are strictly determined by the properties of the enzyme and not by the enzyme-graphite interface. It can also be stated that the decrease in the electrocatalytic activity of the BOD-modified electrodes in the high alkaline region is due to conversion of the active enzyme into an inactive form due to complex formation between T2 Cu2+ and OH [6].
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The obtained results show that the half wave potential for catalytic oxygen electroreduction changes with about 30 mV/pH, whereas the E0 0 (O2/H2O) has a 60 mV/pH dependence. The pH dependence of the T1 site of BOD has not been estimated yet. However, similar results have been demonstrated for the electrocatalytic reduction of O2 by laccase. It was found that the half wave potential for the catalytic oxygen electroreduction by laccases from different origins changes with about 15 mV/pH [32]. A similar pH dependence was previously shown by Nakamura [39] for the redox potential of the T1 site of a low redox potential laccase from Rhus vernicifera and approximately estimated by Reinhammar [40] for a high redox potential laccase from P. versicolor. Moreover, the mentioned similarities of the pH dependencies for the heterogeneous electrocatalysis and the homogeneous characteristics (activity vs. pH, formal potential of the T1 site vs. pH) were also found for laccases from different origins [6,11,32]. Thus, all our data point to the fact that the T1 site is the first electron acceptor in DET from a carbon electrode to the enzyme. The conclusion is based on the fact that the observed dependencies of the electrocatalytic currents on pH for adsorbed BOD closely correlate with those obtained with the corresponding dissolved enzymes for some substrates, where the T1 site is determined to be the first electron acceptor [23,30]. An additional fact that supports this conclusion is that the half wave potential of oxygen electroreduction at graphite electrodes modified with BOD is close to the redox potential of the T1 site of the enzyme when compared with the E0 0 (O2/H2O) under the same conditions. Thus, the conclusion that the T1 site is the first electron acceptor in the enzyme when adsorbed on carbon electrodes under aerobic condition is well motivated. To summarize, all our results with SPGE modified with BOD from M. verrucaria support the possibility of DET between the enzyme and the electrode under aerobic and anaerobic conditions. Moreover, the idea that the T1 site is the first electron acceptor from the electrode in the heterogeneous reduction of the enzyme was confirmed. Our results are in a good agreement with previous data concerning DET processes for other ‘‘blue’’ multicopper oxidases, such as laccase, ascorbate oxidase, and ceruloplasmine [6,8,10,11,32].
Acknowledgements The work was financially supported by the Swedish Research Council, the Russian Foundation for Basic Research (Project No. 03-04-48937), and the Russian Foundation Programme ‘‘Biocatalytic Technologies’’ (Contract No. 43.073.1.1.2505).
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