Comparison of Direct and Mediated Electron Transfer for Cellobiose

Mar 3, 2009 - The midpoint potentials at pH 3.5 for the flavin (40 mV) and the heme domain (170 mV) were determined with spectroelectrochemistry...
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Anal. Chem. 2009, 81, 2791–2798

Comparison of Direct and Mediated Electron Transfer for Cellobiose Dehydrogenase from Phanerochaete sordida Federico Tasca,† Lo Gorton,† Wolfgang Harreither,‡ Dietmar Haltrich,‡ Roland Ludwig,§ and Gilbert No¨ll*,†,| Department of Analytical Chemistry, Lund University, P.O. Box 124, SE-221 00 Lund, Sweden, Division of Food Biotechnology, Department of Food Sciences and Technology, BOKU-University of Natural Recources and Applied Life Sciences Vienna, Muthgasse 18, A-1190 Wien, Austria, Research Centre Applied Biocatalysis, Petersgasse 14, A-8010 Graz, Austria, and Siegen University, Organic Chemistry 1, Adolf-Reichwein-Strasse 2, 57068 Siegen, Germany Direct and mediated electron transfer (DET and MET) between the enzyme and electrodes were compared for cellobiose dehydrogenase (CDH) from the basidiomycete Phanerochaete sordida (PsCDH). For DET, PsCDH was adsorbed at pyrolytic graphite (PG) electrodes while for MET the enzyme was covalently linked to a low potential Os redox polymer. Both types of electrodes were prepared in the presence of single walled carbon nanotubes (SWCNTs). DET requires the oxidation of the heme domain, while MET occurs partially via the heme and the flavin domain at pH 3.5. At pH 6 MET occurs solely via the flavin domain. Most probably, the interaction of the domains decreases from pH 3.5 to 6.0 due to electrostatic repulsion of deprotonated amino acid residues, covering the surfaces of both domains. MET starts at a lower potential than DET. The midpoint potentials at pH 3.5 for the flavin (40 mV) and the heme domain (170 mV) were determined with spectroelectrochemistry. The electrochemical and spectroelectrochemical measurements presented in this work are in conformity. The pH dependency of DET and MET was investigated for PsCDH. The optimum was observed between pH 4 and 4.5 pH for DET and in the range of pH 5-6 for MET. The current densities obtained by MET are 1 order of magnitude higher than by DET. During multicycle cyclic voltammetry experiments carried out at different pHs, the PsCDH modified electrode working by MET turned out to be very stable. In order to characterize a PsCDH modified anode working by MET with respect to biofuel cell applications, this electrode was combined with a Pt-black cathode as model for a membraneless biofuel cell. In comparison to DET, a 10 times higher maximum current and maximum power density in a biofuel cell application could be achieved by MET. While CDH modified electrodes working by DET are * To whom correspondence should be addressed. Gilbert No ¨ll, Siegen University, Organic Chemistry 1, Adolf-Reichwein-Str. 2, 57068 Siegen, Germany. Phone: +49 (0)271 740-4360. Fax: +49 (0) 271 740-3270. E-mail: noell@ chemie.uni-siegen.de. † Lund University. ‡ BOKU-University of Natural Recources and Applied Life Sciences Vienna. § Research Centre Applied Biocatalysis. | Siegen University. 10.1021/ac900225z CCC: $40.75  2009 American Chemical Society Published on Web 03/03/2009

highly qualified for applications in amperometric biosensors, a much better performance as biofuel cell anodes can be obtained by MET. The use of CDH modified electrodes working by MET for biofuel cell applications results in a less positive onset of the electrocatalytic current (which may lead to an increased cell voltage), higher current and power density, and much better longterm stability over a broad range of pH. Cellobiose dehydrogenases (CDHs, EC 1.1.99.18) are extracellular redox enzymes produced by a variety of fungi from the phyla of Basidiomycota and Ascomycota.1-3 CDHs exist usually as monomers built from a single amino acid chain, comprising a larger flavin-associated (dehydrogenase) and a smaller hemebinding (cytochrome) domain. The cofactors in the flavin and heme domain are flavin adenine dinucleotide (FAD) and heme b.1-3 CDHs catalyze the oxidation of di- and oligosaccharides at the C(1) carbon to the corresponding lactones over a broad pH range.1-3 Besides their natural substrates, β-D-cellobiose and cellooligosaccharides, also lactose, having the same β-1,4 glycosyl linkage, are oxidized with high catalytic efficiency by all CDHs. In contrast to CDHs from basidiomycete fungi, the ascomycete CDH from Myriococcum thermophilum (MtCDH) catalyzes also the oxidation of monosaccharides such as glucose and some diand oligosaccharides, e.g., maltose consisting of two R-1,4-linked glucose moieties, with high turnover rates.1,3 CDHs belong to a group of enzymes, which are able to communicate directly with electrodes without the need of any redox mediator.4-7 Because of their catalytic activity, CDHs have been widely used for the (1) Zamocky, M.; Ludwig, R.; Peterbauer, C.; Hallberg, B. M.; Divne, C.; Nicholls, P.; Haltrich, D. Curr. Protein Pept. Sci. 2006, 7, 255–280. (2) Christenson, A.; Dimcheva, N.; Ferapontova, E. E.; Gorton, L.; Ruzgas, T.; Stoica, L.; Shleev, S.; Yaropolov, A. I.; Haltrich, D.; Thorneley, R. N. F.; Aust, S. D. Electroanalysis 2004, 16, 1074–1092. (3) Harreither, W.; Coman, V.; Ludwig, R.; Haltrich, D.; Gorton, L. Electroanalysis 2007, 19, 172–180. (4) Ferapontova, E. E.; Shleev, S.; Ruzgas, T.; Stoica, L.; Christenson, A.; Tkac, J.; Yaropolov, A. I.; Gorton, L. In Electrochemistry of Nucleic Acids and Proteins; Palacek, E., Scheller, F. W., Wang, J., Eds.; Elsevier: Amsterdam, The Netherlands, 2005, pp 517-598. (5) Wollenberger, U. In Biosensors and Modern Biospecific Analytical Techniques; Gorton, L., Ed.; Elsevier: Amsterdam, The Netherlands, 2005, pp 65-130.

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Scheme 1. Schematic Model Showing DET (Left) and MET for CDH (Right)a

a The linker region connecting the N-terminal cytochrome domain to the flavin domain was drawn arbitrarily. During DET, electrons are transferred one-by-one from the reduced FAD to the heme cofactor and from there to the electrode surface (possibly by movement of the heme domain). In the case of MET, the electrons are shuttled by the Os polymer to the electrode.

development of amperometric biosensors working by direct electron transfer (DET).3,8-10 Recently a lactose biosensor based on CDH from basidiomycete fungi working by DET has been presented.9 This sensor is of interest due to its simplicity of construction, experimental characteristics, and substrate specificity (CDH from basidiomycete fungi shows no reaction with glucose or other monosaccharides). The oxidation of a substrate at the flavin domain of CDH is followed by intramolecular (internal) electron transfer (IET) to the heme domain, which is able to donate the electrons to the electrode.3,8-10 Alternatively to DET, the enzymes can be covalently linked to an Os redox polymer. In this case ET is mediated by the flexible Os redox centers, which transfer the electrons from the enzymes to the electrode.2,3 Whereas mediated electron transfer (MET) has been shown for the intact enzyme as well as for the separated flavin domain, DET requires the presence of the heme domain.2,3,11 A schematic model showing the differences between DET and MET is presented in Scheme 1. While the first type of CDH was described already more than 30 years ago,12-14 only now CDHs are getting in the focus of biofuel cell research.15-17 Recently, five different types of CDH were screened with respect to their DET properties after coadsorption with single-walled carbon nanotubes (SWCNTs) on graphite electrodes.15 The CDHs from the basidiomycete fungi (6) Borgmann, S.; Hartwich, G.; Schulte, A.; Schuhmann, W. In Electrochemistry of Nucleic Acids and Proteins; Palacek, E., Scheller, F. W., Wang, J., Eds.; Elsevier: Amsterdam, The Netherlands, 2005, pp 599-655. (7) Le´ger, C.; Bertrand, P. Chem. Rev. 2008, 108, 2379–2438. (8) Stoica, L.; Dimcheva, N.; Haltrich, D.; Ruzgas, T.; Gorton, L. Biosens. Bioelectron. 2005, 20, 2010–2018. (9) Stoica, L.; Ludwig, R.; Haltrich, D.; Gorton, L. Anal. Chem. 2006, 78, 393– 398. (10) Stoica, L.; Ruzgas, T.; Ludwig, R.; Haltrich, D.; Gorton, L. Langmuir 2006, 22, 10801–10806. (11) Larsson, T.; Elmgren, M.; Lindquist, S.-E.; Tessema, M.; Gorton, L.; Tessema, M.; Henriksson, G. Anal. Chim. Acta 1996, 331, 207–215. (12) Westermark, U.; Eriksson, K. E. Acta Chem. Scand., Ser. B 1974, 28, 209– 214. (13) Westermark, U.; Eriksson, K. E. Acta Chem. Scand., Ser. B 1974, 28, 204– 208. (14) Ayers, A. R.; Ayers, S. B.; Eriksson, K. E. Eur. J. Biochem. 1978, 90, 171– 181. (15) Tasca, F.; Gorton, L.; Harreither, W.; Haltrich, D.; Ludwig, R.; No ¨ll, G. J. Phys. Chem. C 2008, 112, 9956–9961. (16) Tasca, F.; Gorton, L.; Harreither, W.; Haltrich, D.; Ludwig, R.; No ¨ll, G. J. Phys. Chem. C 2008, 112, 13668–13673. (17) Coman, V.; Vaz-Domı´nguez, C.; Ludwig, R.; Harreither, W.; Haltrich, D.; De Lacey, A. L.; Ruzgas, T.; Gorton, L.; Shleev, S. Phys. Chem. Chem. Phys. 2008, 10, 6093–6096.

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Phanerochaete sordida (PsCDH), Phanerochaete chrysosporium (PcCDH), and Trametes villosa (TvCDH), from the ascomycete fungus Myriococcum thermophilum (MtCDH), and from the plant pathogen fungus Sclerotium rolfsii (SrCDH) were compared.15 In the presence of oxidatively shortened and length separated SWCNTs, it was even possible to directly observe the reduction of the flavin and the heme domains of PsCDH and MtCDH by cyclic and square wave voltammetry.15 On the basis of PsCDH, which exhibited the highest rate of DET, a biofuel cell anode working by DET was developed.15 Lactose and cellobiose were used as a substrate. In another paper, a biofuel cell anode working by MET based on MtCDH was presented.16 To the best of our knowledge, these two contributions are the first detailed studies on CDH modified electrodes with respect to biofuel cell applications.15,16 Besides lactose and cellobiose, the MtCDH based anode could also oxidize glucose over a broad range of pH with high efficiency.16 Because of their broad range of substrates (including monosaccharides such as glucose), especially some CDHs from ascomycete fungi are of interest not only for applications in biosensors (as long as there is no substrate interference) but also for the development of biofuel cell anodes. When for MtCDH polarization curves using lactose as substrate were measured at neutral pH, the onset of the electrocatalytic current was limited by the redox potential of the low potential Os polymer. Only when the polarization curves were measured at low pH, an additional onset in the catalytic current was detected at a more positive potential.16 This second onset was almost at the same potential, at which DET started when the enzyme was adsorbed in the absence of Os polymer. A possible explanation for this observation is that the additional onset was caused by wiring the heme domain of some enzyme molecules, which have not been able to communicate with the Os polymer via the flavin domain.16 However, a more detailed study was not performed as the rate of DET for MtCDH was rather low. In this contribution, DET and MET for PsCDH modified electrodes are compared in detail. PsCDH was chosen for this study due to its ability to exhibit a high rate of DET.15 The midpoint potentials of the flavin and heme domain of PsCDH were determined by spectroelectrochemistry at pH 3.5. Besides the pH dependency, also the stability of a PsCDH modified electrode working by MET was monitored, and a biofuel cell application is presented.

EXPERIMENTAL PART Equipment and Electrochemical Measurements. Water was purified in a Milli-Q water purification system (Millipore, Bedford, MA). Single-walled carbon nanotubes were purchased from Nanocyl, Sambreville, Belgium. Poly(ethylene glycol) (400) diglycidyl ether (PEGDGE) was obtained from Aldrich (http:// www.sigmaaldrich.com). Poly(vinylpyridine)-[osmium-(N,N′-methylated-2,2′-biimidalzole)3]2+/3+ was synthesized as reported elsewhere.18 Electrochemical measurements were performed with an EG&G potentiostat/galvanostat model 273 A using modified electrodes as the working electrode, a saturated calomel reference electrode (SCE), and a platinum foil counter electrode. Unless otherwise stated, argon was purged through the solutions for some minutes prior to the experiments. All potentials discussed in the main part are referred to the normal hydrogen electrode (NHE). Homemade pyrolytic graphite electrodes with a squared surface of 3 mm × 3 mm were used. Pyrolytic graphite was obtained as a gift from Mr. Robert Pulley, Minerals Technologies (mineralstech.com). The current densities were calculated with respect to the geometric electrode area. Enzyme-modified electrodes were prepared by adsorbing the enzyme solution (10 µL) in the presence of 10-15 µL of a suspension of SWCNTs, (10 mg mL-1). The SWCNT suspension was made by overnight sonication of the SWCNTs in Milli-Q water. For MET, additionally 6-8 µL of an Os redox polymer solution (10 mg mL-1) were added and 2-4 µL of poly(ethylene glycol) (400) diglycidyl ether PEGDGE (35 mg mL-1) were gently mixed to the drop at the electrode surface. The electrodes were then allowed to dry overnight at 4 °C. All solutions used for immobilization were prepared in Milli-Q water. There was some variation in the absolute activity of individual electrodes, because the suspension of SWCNTs used for electrode modification was not completely homogeneous (even after extended sonication), and the amount of adsorbed SWCNTs may vary. The activity of PsCDH (EC 1.1.99.18) was 140 U mL-1 (specific activity 33 U mg-1), and the cultivation and purification of the enzyme has been described previously.15 Spectroelectrochemistry. The spectroelectrochemical setup used in this work has been reported elsewhere.19 As redox mediators,20-22 2,6-dichloroindophenol, phenazine methosulfate, methylene blue, resazurin, 2-OH-1,4-naphthoquinone, anthraquinone 2,6-disulfonate, safranine T, diquat, 1,1′-bis(hydroxyethyl)-4,4′bipyridyl dichloride, and 1,1′-propylene-2,2′-bipyridylium dibromide were applied at a concentration of about 25 µM. At this concentration, the electrochemical equilibrium can be established in a reasonable period after changing the potential, and at the same time, the contribution of the mediators to the optical spectra in the range between 400 and 500 nm is only minor. Spectrophotometric Measurements. Spectrophotometric measurements of the pH dependent activity of PsCDH in solution were performed by using the one-electron acceptor cytochrome c and the two-electron-proton acceptor 2,6-dichloroindophenol (18) Mao, F.; Mano, N.; Heller, A. J. Am. Chem. Soc. 2003, 125, 4951–4957. (19) Bistolas, N.; Christenson, A.; Ruzgas, T.; Jung, C.; Scheller, F. W.; Wollenberger, U. Biochem. Biophys. Res. Commun. 2004, 314, 810–816. (20) Dutton, P. L. Methods Enzymol. 1978, 54, 411–435. (21) Wilson, G. S. Methods Enzymol. 1978, 54, 396–410. (22) No ¨ll, G.; Hauska, G.; Hegemann, P.; Lanzl, K.; No ¨ll, T.; von Sanden-Flohe, M.; Dick, B. ChemBioChem 2007, 8, 2256–2264.

(DCIP) in 0.1 M sodium citrate buffer between pH 2.9 and 6.5 and in 0.1 M sodium phosphate buffer between pH 6.5 and 8.0 following established methods.23 Examination of the PsCDH Modified Electrodes for a Membraneless Biofuel Cell Application. A PsCDH modified electrode was applied as the anode together with a Pt black cathode in 0.1 M lactose solution (0.1 M phosphate buffer, pH 6) as a model for a membraneless biofuel cell. Pt black electrodes were made by galvanostatic adsorption of Pt to Pt electrodes from a plating solution containing 25 mM hydrochloric acid, in which 3% (w/v) platinum(IV) chloride and 0.025% (w/v) lead acetate was dissolved. The Pt electrodes consisted of a coiled Pt foil (∼7 mm × 20 mm) melted to a Pt wire or of a Pt spiral with a diameter of 1 mm and a length of about 40 mm. The ends of the Pt wires were sealed in a glass tube. The surfaces of the Pt black electrodes used as cathodes were several times larger than that of the anodes in order to (i) limit the maximum cell current by the anode (not by the cathode) and (ii) prevent any decrease in current density with time due to possible passivation of the cathode. Polarization curves were measured by linear sweep voltammetry connecting the anode as working, and the cathode as the reference and counter electrodes.15,16 In order to avoid any contribution of capacitive current, the applied scan rate was very low (0.1 mV s-1). Furthermore, we scanned from 0 to -600 mV, i.e., from zero to the maximum cell voltage. In this case, any capacitive current would add to the pristine polarization curve with a negative sign. Since the polarization curve was determined by linear sweep voltammetry, it is plotted as J vs E. RESULTS AND DISCUSSION Comparison of DET and MET by Cyclic Voltammetry. In Figure 1A the cyclic voltammograms (CVs) of a PsCDH modified pyrolytic graphite (PG) electrode working by DET at pH 3.5 in the presence and absence of substrate (0.1 M lactose) are shown. The electrocatalytic current for lactose oxidation starts at about 100 mV. As proven by control experiments, the small wave at ∼-40 mV is caused by FAD released from the protein. In Figure 1B the CVs of a PsCDH/SWCNT/Os polymer modified PG electrode working by MET at pH 3.5 in the presence and absence of substrate (0.1 M lactose) are presented. In the absence of substrate, the oxidation/reduction of the Os polymer can be seen at 40 mV. In the presence of substrate, the electrocatalytic current starts at -20 mV. A second onset in the electrocatalytic current can be seen at about 110 mV, at a similar potential as the onset for DET shown in Figure 1A. A similar behavior was reported earlier for MtCDH.16 In line with this previous study, we suggest that at -20 mV the Os polymer starts to collect electrons gained by substrate oxidation from the flavin domain. Obviously, not all enzyme molecules are able to communicate with the Os polymer via their flavin domain at pH 3.5. A possible explanation is that at pH 3.5 the flavin and the heme domain are located closely together, and for some enzyme molecules the heme domain is blocking the direct access of the Os redox centers to the active site of the flavin domain. The situation is different at pH 6 (see Figure 1C). At pH 6 there is a larger distance between the flavin and heme domain, and for all enzyme (23) Baminger, U.; Subramaniam, S. S.; Renganathan, V.; Haltrich, D. Appl. Environ. Microbiol. 2001, 67, 1766–1774.

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Figure 1. CVs of a PsCDH/SWCNT modified PG electrode at pH 3.5 (0.1 M citrate buffer) in the presence and absence of substrate (0.1 M lactose). (B) CVs of a PsCDH/SWCNT/Os polymer modified PG electrode at pH 3.5 (0.1 M citrate buffer) in the presence and absence of substrate (0.1 M lactose). (C) Linear sweep voltammograms of a second PsCDH/SWCNT/Os polymer modified PG electrode at pH 3.5 (0.1 M citrate buffer) and pH 6 (0.1 M phosphate buffer) in the presence and absence of substrate (0.1 M lactose). All voltammograms were measured at a scan rate of 1 mV s-1.

molecules the flavin domain can be addressed directly by the Os polymer. Already in previous studies of CDH modified electrodes with respect to biosensor applications, a change in geometry with increasing pH was suggested.9,10 It was observed that DET, which requires IET from the flavin to the heme domain, was most efficient at pH values close to pH 4.5 (depending on the individual types of CDH).8-10 This led to the assumption that the pH for optimum IET is in between the isoelectric points of the individual domains.10 At this pH, both domains are oppositely charged and the ET distance is minimized due to electrostatic attraction, whereas at pH 6 the interaction of the domains decreases due to electrostatic 2794

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Figure 2. (A) Linear sweep voltammograms of a second PsCDH/ SWCNT modified PG electrode measured at a scan rate of 1 mV s-1 from pH 4 to 6 in the presence of 0.1 M lactose. In part B, the pH profile for PsCDH in solution determined by a spectrophotometric assay using cytochrome c as an electron acceptor is shown. The electrons are passed from the flavin domain via the heme domain to this electron acceptor. The activity is thus influenced by the intramolecular electron transfer (IET) between the two domains and resembles the ET pathway in the DET mode. In part C, the pH profile for PsCDH in solution determined by a spectrophotometric assay using DCIP as a two-electron--proton acceptor is depicted. The flavin domain can donate electrons directly to this two-electron-proton acceptor resembling the ET pathway in the MET mode.

repulsion of deprotonated amino acid residues, covering the surfaces of both domains. Also in the current study, the pH dependency of DET was studied (see Figure 2A). DET was most efficient between pH 4 and 4.5 (with slightly higher current observed for pH 4.5 than for pH 4), whereas MET had a maximum between pH 5 and 6 (not shown). At neutral pH, MET was still quite efficient, while DET was almost completely switched off by lack of IET. As depicted in parts B and C of

Figure 2, the activities of PsCDH were measured also by a spectrophotometric assay using the electron acceptors cytochrome c and DCIP resembling the ET pathway in the DET and MET mode, respectively (in contrast to DCIP, cytochrome c does not accept electrons from the separate flavin domain of CDH).1 The pH optima derived from the spectrophotometric measurements are by a value of 0.5 lower than those for the electrochemical measurements but follow the same tendency and show a higher pH optimum for the flavin domain’s activity than for the IET dependent process involving the heme domain. The small deviation is probably caused by a repulsive effect between the electron acceptors (cytochrome c or DCIP) and the increasingly stronger negative force field of PsCDH at pH values above its isoelectric point of 4.0. It can be seen from Figure 1C that MET at pH 6 starts much earlier (at about -100 mV) than at pH 3.5 (at -20 mV). This implies that at low pH, the onset of the electrocatalytic current is limited by the midpoint potential of the flavin domain but not by the midpoint potential of the Os polymer, the latter being almost independent of pH.24 For free flavins following a two-electronone-proton reduction, a shift of -59 mV per pH unit is expected.25,26 This is in accordance with the pH dependency observed for the reduction of the heme domain of CDH from Phanerochaete chrysosporium (PcCDH).27 For PcCDH, the midpoint potentials of the flavin (106 mV, pH 3/-132 mV, pH 7) and the heme domain (190 mV, pH 3/130 mV, pH 7) were determined by redox titration.27 Also the reduction of the heme domain was found to be pH dependent, because some residues close to the heme change their protonation state due to different pKa values in the reduced and oxidized forms of CDH.28 This can also be seen from Figure 2. The catalytic current for lactose oxidation starts at slightly more negative values when the pH is increased. Spectroelectrochemistry. In order to determine the midpoint potentials of the flavin and the heme domain of PsCDH at pH 3.5, spectroelectrochemical measurements were carried out. In Figure 3A the spectral changes during the complete reduction of PsCDH are shown. From the experimental data, a midpoint potential of 40 mV for the flavin and 170 mV for the heme domain were determined. There might be a minor experimental error (up to ±20 mV), because the experiments were carried out in the presence of redox mediators, which contribute to the absorption spectra depending on their redox states.20-22 The presence of mediators was required in order to establish electrochemical equilibrium. Within the experimental error, the same midpoint potentials were measured during reduction and reoxidation. According to Nernst (E ) E°′ + 0.059 V/n log(COx/CRed)), at 50 mV the reduction of the heme (n ) 1) domain was almost complete (>99% turnover). The reduction of the flavin (n ) 2) started at 100 mV, and at 70 mV about 10% of the flavins were reduced. Hence, at pH 3.5 the reductions of both domains are overlapping. This is obvious (24) Heller, A. Phys. Chem. Chem. Phys. 2004, 6, 209–216. (25) No ¨ll, G.; Kozma, E.; Grandori, R.; Carey, J.; Scho ¨dl, T.; Hauska, G.; Daub, J. Langmuir 2006, 22, 2378–2383. (26) Clark, W. M. Oxidation-Reduction Potentials of Organic Systems; Williams & Wilkins Co.: Baltimore, MD, 1960. (27) Igarashi, K.; Momohara, I.; Nishino, T.; Samejima, M. Biochem. J. 2002, 365, 521–526. (28) Lindgren, A.; Gorton, L.; Ruzgas, T.; Baminger, U.; Haltrich, D.; Schulein, M. J. Electroanal. Chem. 2001, 496, 76–81.

Figure 3. Spectroelectochemistry of PsCDH at pH 3.5 (0.1 M citrate buffer, 0.1 M KCl) measured in the presence of a mixture of redox mediators. The concentration of the individual mediators was about 25 µM. Depending on their redox states, the mediators are contributing to the optical spectra to some extent (the change in absorption between 600 and 700 nm is mainly caused by the mediator mixture). In part A, the complete set of spectra collected during ongoing reduction is shown. In parts B and C, the spectra representing mainly the first and second reduction step are depicted. The missing absorption in the spectra around 665 nm is an error of the instrument caused by the deuterium light source.

from the spectra depicted in Figure 3B,C, which mainly represent the first and second reduction step. Before the reduction of the heme domain indicated by increasing bands at 429 and 563 nm was complete, the reduction of the flavin domain, monitored by a decrease in absorption between 440 and 500 nm, started. The reduction of the flavin domain was a two-electron-one-proton reduction following an “ece” mechanism22 (electrochemical step, chemical step/protonation, electrochemical step) without formation of a stable negatively charged flavosemiquinone radical anion or a neutral flavosemiquinone radical within the time scale of the Analytical Chemistry, Vol. 81, No. 7, April 1, 2009

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experiment.22,29 With dependence on the pKa of the doubly reduced neutral flavohydroquinone, a second protonation step may follow the second ET step leading to the flavohydroquinone in its neutral form. The spectra collected in this work differ from those presented for the reduction of MtCDH.30 It seems that only the heme domain of MtCDH could be reduced completely when the whole enzyme was studied in the absence of redox mediators.30 The midpoint potentials determined for PsCDH at pH 3.5 in this work are in good agreement with an electrochemical investigation monitoring directly the reduction of the flavin and the heme domain at pH 4.0 by cyclic and square wave voltammetry.15 Furthermore, the electrochemical and spectroelectrochemical measurements presented in this work are in conformity. At the potentials of 40 and 170 mV, determined as midpoint potentials for the flavin and the heme domain by spectroelectrochemistry, the increase in catalytic current (i.e., the slope of the curve) measured by cyclic voltammetry (see Figure 1B) is at its maximum. This behavior is expected if the Os polymer is not limiting the potential for substrate oxidation. In Figure 1B, the electrocatalytic current starts at -20 mV, i.e., with the beginning oxidation of the flavin domain. At about 100 mV, the oxidation of the flavin domain is complete (>99%) and the electrocatalytic current has almost reached a first maximum. Close to this value (at about 110 mV), the second onset of the electrocatalytic current starts, because the heme domain starts to be oxidized and to deliver electrons from the flavin domain to the Os polymer. To some extent, also DET from the heme domain to SWCNTs or to the PG electrode might contribute to the second onset of the electrocatalytic current. At pH 6, the midpoint potential of the flavin domain is expected to be about -110 mV (according to a shift of -59 mV per pH unit from the midpoint potential of 40 mV at pH 3.5). The onset of the catalytic current at pH 6 starts at about -100 mV with the beginning of the oxidation of the Os polymer (at -100 mV somewhat less than 1% of the Os redox centers are oxidized). Hence, at pH 6 the onset of the electrocatalytic current is limited by the oxidation of the Os polymer, whereas at pH 3.5 the potentials for the oxidation of the flavin and heme domain are limiting the electrocatalytic current as indicated by the two onsets in the CV shown in Figure 1B. Stability Measurements. For a PsCDH/SWCNT/cross-linker modified electrode working by DET, a decrease in catalytic current for lactose oxidation of about 20% within 12 h was observed.15 In order to check the stability of a PsCDH modified electrode working by MET, multicycle CVs were recorded at different pH (pH 6-3.5) using lactose as a substrate. At each pH value, 12 cycles with a scan rate of 0.2 mV s-1 were measured (the duration of one multicycle CV was 20 h). Within the first multicycle CV collected at pH 6, the capacity of the modified electrode (notified by the difference in current during the forward and backward scan) decreased and the current response became more consistent. Possibly some structural reorganization in the material at the electrode had taken place within the first 20 h of performance. Before proceeding with a (29) No ¨ll, G. J. Photochem. Photobiol., A 2008, 200, 34–38. (30) Coman, V.; Harreither, W.; Ludwig, R.; Haltrich, D.; Gorton, L. Chem. Anal. (Warsaw, Pol.) 2007, 52, 945–960.

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Figure 4. Multicycle CVs (12 cycles each) of a PsCDH modified electrode working by MET at different pHs starting from pH 6 (A). As substrate 0.1 M lactose was chosen, and a scan rate of 0.2 mV s-1 was applied. The duration of one multicycle CV was 20 h. After 1 week in buffer solution and 100 h of performance, an additional multicycle CV was measured at pH 6. The first and last multicycle CVs are depicted in part B. The total decrease in activity was about 33%.

new multicycle CV at the next pH value, the electrode was kept in buffer solution for some additional hours. The set of five multicycle CVs presented in Figure 4A was measured within a period of 1 week. After 100 h of performance and being stored in different buffer solutions for 1 week in total, an additional multicycle CV for the same electrode was measured at pH 6 (see Figure 4B). The total decrease in activity was only about 33%. Hence, the PsCDH modified electrode working by MET was much more stable than a PsCDH modified electrode working by DET, which has been described previously.15 Also in the multicycle CVs collected for pH 3.5-4.5, the additional onset in catalytic current for wiring the heme domain as discussed previously can be seen. Biofuel Cell Performance. Recently, a PsCDH/SWCNT/ cross-linker modified electrode working by DET was studied with respect to biofuel cell applications.15 The PsCDH modified electrode was applied as an anode together with a Pt black cathode in a 5 mM lactose solution (at pH 4.5, citrate buffer, 0.1 M) as model for a membraneless biofuel cell working by DET at both electrodes.15 After operating for about 1 h, a polarization curve was measured by linear sweep voltammetry (v ) 0.2 mV s-1) connecting the anode as working, and the cathode as the reference and counter electrodes. The cell exhibited a maximum voltage of 590 mV, a maximum current density of 112 µA cm-2, and a maximum power density of 32 µW cm-2 at an operating voltage of 430 mV (under oxygen purging/non-

mA cm-2, and a maximum power density of 300 µW cm-2 at an operating voltage of 350 mV (under oxygen purging/ nonquiescent conditions). A fill factor of 0.40 was calculated. Since both experiments were carried out at different pHs (DET, pH 4.5; MET, pH 6) and also the potential of oxygen reduction is pH dependent,31-33 the maximum cell voltage for MET was not increased in comparison to DET, even though during MET the oxidation of the substrate begins at a less positive potential. In comparison to DET,15 a 10 times higher maximum current and maximum power density in a biofuel cell application can be achieved by MET, when PsCDH is used as the catalyst at the anode. This tendency was already indicated by comparing the current densities for lactose oxidation in the CVs depicted in Figure 1 for DET and MET. The maximum power density obtained for the PsCDH MET anode/Pt cathode biofuel cell was twice as high as reported for a glucose biofuel cell comprising a MtCDH anode working by MET at pH 7.4 and a Pt cathode.16

Figure 5. (A) Polarization curve (measured with linear sweep voltammetry by scanning from 0 to -600 mV with a scan rate of 0.1 mV s-1 after an equilibration time of 10 min) and (B) dependence of the power density on the operating voltage for a membraneless biofuel cell consisting of a PsCDH/SWCNT/Os polymer modified PG electrode as the anode and a Pt black electrode as the cathode. As fuel, a 0.1 M lactose solution (0.1 M phosphate buffer, pH 6) was used.

quiescent conditions).15 A fill factor of 0.48 was calculated. It was suggested that by replacing the Pt cathode by a laccase or bilirubin oxidase modified cathode, the maximum voltage of the cell can be further increased.15 This has been shown by a combination of a new type of CDH from the ascomycete Dichomera saubinetii (DsCDH) as catalyst at the anode with the well-characterized laccase from Trametes hirsuta at the cathode.17 DsCDH was even able to oxidize glucose, but the highest power density of 15 µW cm-2 and a maximum open circuit potential (i.e., a maximum cell voltage) of 770 mV were measured with lactose as a substrate at pH 4.5.17 In order to characterize a PsCDH modified anode working by MET with respect to biofuell cell applications, this electrode was combined with a Pt-black cathode as a model for a membraneless biofuel cell (analogue to the PsCDH/SWCNT/cross-linker modified electrode working by DET). Since the surface of the Pt-black cathode was much larger than that of the anode and oxygen was purged gently around the cathode, the maximum current density was limited by the anode. Lactose (0.1 M) was used as the substrate, and the experiment was performed at pH 6 (close to neutral pH). After an equilibration time of 10 min, a polarization curve was measured by linear sweep voltammetry (v ) 0.1 mV s-1) connecting the anode as the working electrode and the cathode as the reference and counter electrodes scanning from 0 to -600 mV. As shown in Figure 5, the cell exhibited a maximum voltage of 570 mV, a maximum current density of 1.32

CONCLUSIONS In this work, DET and MET for PsCDH modified anodes were compared. In line with previous investigations,8-10,15 it was observed that DET requires the oxidation of the heme domain. When MET was investigated at pH 3.5 by cyclic voltammetry using lactose as the substrate, an additional onset of the electrocatalytic current was observed, which can be explained as follows. At low pH, the flavin domain of some enzyme molecules can be addressed directly by the Os redox centers, while for others the oxidation of the heme domain is required. By changing the pH to less acidic values, Os redox centers become able to collect the electrons directly from the flavin domain for all enzyme molecules. A possible reason for this behavior is a pH dependent change of the orientation of the flavin and the heme domain relative to each other.10 For PsCDH, the optimum pH range for DET is pH 4-4.5 and for MET pH 5-6. MET starts at an up to 200 mV less positive redox potential than DET. Even though PsCDH exhibits a relative high rate of DET compared to other CDHs,15 the current densities, which can be achieved by MET, are still 1 order of magnitude higher than by DET. Furthermore, PsCDH modified electrodes working by MET are much more stable than those working by DET. Most of the advantages of PsCDH modified electrodes working by MET in comparison to DET presented in the current study, such as a less positive onset of the electrocatalytic current, higher activity at less acidic or neutral pH, higher current density, and higher stability, have been reported previously for MtCDH3,16 and can be expected also for further types of CDH. These aspects are of utmost importance when possible applications of CDHs are discussed. The use of CDH modified electrodes working by DET is extremely useful for the development of amperometric biosensors, which have to be cheap and are designed for fast response time and single use. The development of a highly efficient lactose biosensor based on a PsCDH modified electrode working by DET has been reported already.9 If the activity of new types of CDH from ascomycete fungi such (31) Walch, S.; Dhanda, A.; Aryanpour, M.; Pitsch, H. J. Phys. Chem. C 2008, 112, 8464–8475. (32) Sepa, D. B.; Vojnovic, M. V.; Vracar, L. M.; Damjanovic, A. Electrochim. Acta 1987, 32, 129–134. (33) Wilshire, J.; Sawyer, D. T. Acc. Chem. Res. 1979, 12, 105–110.

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as MtCDH3,16 or DsCDH17 toward oxidation of glucose by DET at physiological pH was sufficiently high, CDH modified electrodes could replace the currently used glucose biosensors consisting of glucose oxidase covalently linked to an Os redox polymer. The development of a glucose sensor working by DET is of great economical interest because it would be cheap and the production of Os containing waste, which is environmentally harmful, could be avoided. The situation is different when it comes to the development of biofuel cell anodes. The main obstacle for any biofuel cell application up to now are low current density and lack of stability but not the use of Os redox polymers. In fact, the most effective type of biofuel cell, which is close to an application as an implantable power source, is based on glucose oxidase at the anode and bilirubin oxidase at the cathode, both enzymes covalently linked to Os redox polymers.24,34-36 When CDH (34) Heller, A. Curr. Opin. Chem. Biol. 2006, 10, 664–672. (35) Barton, S. C.; Gallaway, J.; Atanassov, P. Chem. Rev. 2004, 104, 4867– 4886. (36) Moehlenbrock, M. J.; Minteer, S. D. Chem. Soc. Rev. 2008, 37, 1188– 1196.

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modified electrodes shall be used as biofuel cell anodes, much better performance can be obtained by MET compared to DET. As shown in this study, the use of CDH modified electrodes working by MET for biofuel cell applications results in a less positive onset of the electrocatalytic current (which may lead to an increased cell voltage), higher current and power density, and much better long-term stability over a broad range of pH.

ACKNOWLEDGMENT This work was supported by the Deutsche Forschungsgemeinschaft DFG (DFG Postdoctoral Fellowship NO 740/1-1 and Ru¨ckkehrstipendium No. 740/3-1), the Swedish Research Council (Projects 621-2004-4476 and 621-2007-4124), and the Austrian Science Fund (Project L395-B11).

Received for review January 30, 2009. Accepted February 5, 2009. AC900225Z