Spectroelectrochemical Investigation of a Flavoprotein with a Flavin

D-93051 Regensburg, Germany, Faculty of Chemistry and Chemical Engineering, Babes-Bolyai UniVersity,. Arany Janos Street No. 11, Cluj_Napoca 3400 ...
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Langmuir 2006, 22, 2378-2383

Spectroelectrochemical Investigation of a Flavoprotein with a Flavin-Modified Gold Electrode Gilbert No¨ll,*,† Erika Kozma,‡ Rita Grandori,§ Jannette Carey,⊥ Thomas Scho¨dl,| Gu¨nter Hauska,| and Jo¨rg Daub† Institut fu¨r Organische Chemie, UniVersita¨t Regensburg, UniVersita¨tsstrasse 31, D-93051 Regensburg, Germany, Faculty of Chemistry and Chemical Engineering, Babes-Bolyai UniVersity, Arany Janos Street No. 11, Cluj_Napoca 3400, Romania, Institute of Chemistry, Johannes Keppler UniVersity, Altenbergerstrasse 69, A-4040 Linz, Austria, Department of Chemistry, Princeton UniVersity, Washington Road, Princeton, New Jersey 08544-1009, and Institut fu¨r Botanik, UniVersita¨t Regensburg, UniVersita¨tsstrasse 31, D-93053 Regensburg, Germany ReceiVed May 31, 2005. In Final Form: October 14, 2005 A flavin-modified gold electrode was developed in order to catalyze the electrochemical oxidoreduction of flavoproteins. Surface modification was carried out by a two-step procedure. In the first step a mixed self-assembled monolayer obtained by adsorption of activated and nonactivated 3,3′-dithiopropionic acid (free acid and N-succinimidyl ester) was formed, followed by the covalent attachment of a N(10)-hexylamino-alkylated flavin derivative via an amide bond in the second step. The electrochemical properties of the flavin-modified electrode are presented and discussed. The redox potential of the attached flavin was measured at various pH values and the electron-transfer rate constant between electrode and flavin was determined as k0 ) 5 s-1 independent of pH. The flavin-modified electrode was successfully applied to the electrochemical and spectroelectrochemical investigation of the flavoprotein WrbA from Escherichia coli that shows some structural similarities to flavodoxins. It is concluded that the electron transfer “electrode f flavin f flavoprotein” occurs by a two-step hopping mechanism where the first step is rate determining. Kinetic details are discussed. Furthermore, it turned out that, in contrast to flavodoxins, where the semiquinone state is stabilized, WrbA rapidly takes up two electrons, directly leading to the fully reduced form. The presented electrode surface modification may generally lend itself for spectroelectrochemical investigations of flavoproteins.

Introduction Spectroelectrochemical investigations allow the kinetic and mechanistic elucidation of electron-transfer (ET) reactions but they are difficult to apply to redox proteins. Because the cofactor is shielded by the protein scaffold, it cannot approach the electrode surface closely during electrochemical measurements. According to Marcus theory,1 the probability for ET decreases exponentially with distance. Therefore, in most cases, direct ET between electrode and cofactor is very slow or not possible at all. Additional problems in protein electrochemistry arise due to slow protein diffusion constants, release of a cofactor, and adsorption at the electrode surface followed by denaturation. Proton-transfer reactions are often coupled with ET which leads to a strong pH dependency of the redox potential. To enhance the ET rate, small electrochemically active molecules, ET mediators, that serve as electron shuttles are often added in low concentration.2 A more controlled way is to link mediator-like moieties at the electrode surface or even at the protein via flexible spacers.3-6 They may shorten the distance to the cofactor and thereby enhance the ET probability. * To whom correspondence should be addressed. Phone: +49 941 943 4610. Fax: +49 941 943 4984. E-mail: gilbert.noell@ chemie.uni-regensburg.de. † Institut fu ¨ r Organische Chemie, Universita¨t Regensburg. ‡ Babes-Bolyai University. § Johannes Keppler University. ⊥ Princeton University. | Institut fu ¨ r Botanik, Universita¨t Regensburg. (1) Marcus, R. A. Angew. Chem. 1993, 105, 1161-1172 (See also: Angew. Chem., Int. Ed. Engl. 1993, 32, 1111-1121). (2) Dutton, P. L. Methods Enzymol. 1978, 54, 411-435. (3) Willner, I.; Katz, E. Angew. Chem., Int. Ed. 2000, 39, 1181-1218. (4) Narvaez, A.; Dominguez, E.; Katakis, I.; Katz, E.; Ranjit, K. T.; Ben-Dov, I.; Willner, I. J. Electroanal. Chem. 1997, 430, 227-233. (5) Zayats, M.; Katz, E.; Willner, I. J. Am. Chem. Soc. 2002, 124, 2120-2121.

A very interesting family of ubiquitous redox-active proteins are the flavoproteins.7-9 They function as oxidoreductases in central metabolic pathways, in the biological ET chains of respiration and photosynthesis, as well as in the formation and cleavage of disulfide bonds. Furthermore, numerous photobiological roles of flavins are known, from the repair of UV-damaged DNA10,11 to blue-light photoreceptors.12-14 At neutral pH, free flavin cofactors undergo an overlapping two-electron, one-proton reduction, where the second reduction potential is more positive than the first.15-18 In contrast, flavodoxins, a highly reductive group of flavoproteins, stabilize the monoreduced flavin state by interaction with the binding site.19 Recently, flavodoxin has been (6) Zayats, M.; Katz, E.; Willner, I. J. Am. Chem. Soc. 2002, 124, 1472414735. (7) Mueller, F., Ed. Chemistry and Biochemistry of FlaVoenzymes, Vol. II; CRC: Boca Raton, FL, 1991. (8) Mueller, F., Ed. Chemistry and Biochemistry of FlaVoenzymes, Vol. I; CRC: Boca Raton, FL, 1991. (9) Hemmerich, P.; Veeger, C.; Wood, H. C. S. Angew. Chem. 1965, 77, 699-716. (10) Ghisla, S.; Massey, V. Eur. J. Biochem. 1989, 181, 1-17. (11) Sancar, A.; Lindsey-Boltz, L. A.; Uensal-Kacmaz, K.; Linn, S. Annu. ReV. Biochem. 2004, 73, 39-85. (12) Schleicher, E.; Kowalczyk, R. M.; Kay, C. W. M.; Hegemann, P.; Bacher, A.; Fischer, M.; Bittl, R.; Richter, G.; Weber, S. J. Am. Chem. Soc. 2004, 126, 11067-11076. (13) Kottke, T.; Dick, B.; Fedorov, R.; Schlichting, I.; Deutzmann, R.; Hegemann, P. Biochemistry 2003, 42, 9854-9862. (14) Crosson, S.; Rajagopal, S.; Moffat, K. Biochemistry 2003, 42, 2-10. (15) Butterfield, S. M.; Goodman, C. M.; Rotello, V. M.; Waters, M. L. Angew. Chem., Int. Ed. 2004, 43, 724-727. (16) Cuello, A. O.; McIntosh, C. M.; Rotello, V. M. J. Am. Chem. Soc. 2000, 122, 3517-3521. (17) Niemz, A.; Imbriglio, J.; Rotello, V. M. J. Am. Chem. Soc. 1997, 119, 887-892. (18) Pellett, J. D.; Becker, D. F.; Saenger, A. K.; Fuchs, J. A.; Stankovich, M. T. Biochemistry 2001, 40, 7720-7728. (19) Astuti, Y.; Topoglidis, E.; Briscoe, P. B.; Fantuzzi, A.; Gilardi, G.; Durrant, J. R. J. Am. Chem. Soc. 2004, 126, 8001-8009.

10.1021/la051423n CCC: $33.50 © 2006 American Chemical Society Published on Web 01/27/2006

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Figure 1. Stepwise modification of the electrode surface by adsorption of mixed disulfides of activated and nonactivated carboxylic acid followed by flavin immobilization via an amide bond.

investigated by electrochemical and spectroelectrochemical methods after immobilization on a transparent tin dioxide electrode.19 Since adsorption may change properties, it remained desirable to establish an experimental setup that allows the investigation of flavoproteins in solution. In 1996 Heering and Hagen reported on the complex electrochemistry of flavodoxin on a glassy carbon electrode.20 They figured out that the reduction of the entire flavodoxin was mediated by flavin mononucleotid (FMN) that was partially released from the protein. Inspired by this observation we present a very suitable flavin-coated gold electrode that is able to catalyze the reduction of flavoproteins in solution. The ET rate between electrode and attached flavin is determined by applying the Laviron approach.21 The present paper concentrates on the description of this flavin-modified electrode and its applicability for the spectroelectrochemical investigation of a flavoprotein. We choose WrbA [tryptophan (W) repressor binding protein A]22-24 that was available to us as an expression protein at high optical density. WrbA has been identified as an Escherichia coli (E. coli) stationary-phase protein that copurifies and co-immunoprecipitates with the tryptophan repressor.22 WrbA has obvious homologues in bacteria, yeasts, fungi, and plants. This protein family displays low but structurally significant sequence similarities to flavodoxins.23 WrbA is a multimer in solution that noncovalently binds one FMN per monomer.24 However, a direct involvement of WrbA in tryptophan regulation still awaits conclusive experimental support and a large set of data rather implicates the WrbA family in the cellular response to oxidative stress. A separate account will detail our progress in characterization of this flavoprotein with respect to flavodoxin (manuscript in preparation). Materials and Methods Purification of WrbA. WrbA was purified as already described24 except for the affinity-chromatography step (Affi-Gel Blue, Biorad), which was replaced by gel filtration (Superdex 75, Amersham Biosciences) in order to preserve the native cofactor. (20) Heering, H. A.; Hagen, W. R. J. Electroanal. Chem. 1996, 404, 249-260. (21) Laviron, E. J. Electroanal. Chem. 1979, 101, 19-28. (22) Yang, W.; Ni, L.; Somerville, R. L. Proc. Natl. Acad. Sci. U.S.A. 1993, 90, 5796-800. (23) Grandori, R.; Carey, J. Protein Sci. 1994, 3, 2185-2193. (24) Grandori, R.; Khalifah, P.; Boice, J. A.; Fairman, R.; Giovanielli, K.; Carey, J. J. Biol. Chem. 1998, 273, 20960-20966.

Electrochemical Setup. Cyclic voltammetry (CV) measurements were performed at room temperature using a undivided electrochemical cell with a three-electrode arrangement and a computercontrolled EG&G potentiostat/galvanostat Model 283 A. As working electrode we used a homemade gold electrode consisting of a cylindrical piece of gold with a diameter of 4 mm sealed in Kel-F. The pH-dependent measurements of the flavin-modified electrode have been carried out in a conventional cell (Metrohm) for solvent volumes of 5-10 mL with a SCE reference and a platinum counter electrode. The potential of the SCE reference electrode is -240 mV vs NHE at room temperature (RT). Investigation of WrbA required a homemade cell constructed for the investigation of small sample volumes, as described elsewhere.25 For this setup the same working electrode was used together with a Ag/AgCl reference and a platinum counter electrode. An Ag/AgCl reference electrode with an outer diameter of 4 mm was purchased from AMETEK. The inner element is a silver wire immersed in a solution of AgClsat./KCl. The potential of this electrode is about -240 mV vs NHE at RT when a 1 M KCl solution is used. Argon was bubbled through all solutions for some minutes before starting the measurements. The supporting electrolyte Na2SO4‚10H2O (MicroSelect) was purchased from Fluka. Distilled water was further purified by a Millipore Mill-Q plus 185 system. The spectroelectrochemical cell applied in this work has been described already in detail.26 A schematic illustration is given in the Supporting Information. A protocol of the electrode surface modification procedure and the synthesis of the flavin cofactor is also listed in the Supporting Information. Furthermore, spectroelectrochemical properties of CofC6 are presented.

Results and Discussion Strategy for Electrode Surface Modification. We synthesized a flavin derivative which can be linked to the electrode via a flexible alkyl chain in order to enhance the ET between electrode and protein. The synthetic cofactor was prepared in such a way that attachment to the electrode by a sulfur-gold bond is possible following a two-step modification procedure shown in Figure 1. Stine et al. reported on the electrochemical behavior of a synthetic flavin sulfide derivative, which was directly adsorbed on gold.27 They examined by microscopic techniques that up to 15% of the gold surface remained uncovered. Furthermore, they could not (25) Smith, E. T.; Bennett, D. W.; Feinberg, B. A. Anal. Chim. Acta 1991, 251, 27-33. (26) Salbeck, J. Anal. Chem. 1993, 65, 2169-2173. (27) Stine, K. J.; Andrauskas, D. M.; Khan, A. R.; Forgo, P.; D’Souza, V. T.; Liu, J.; Friedman, R. M. J. Electroanal. Chem. 1999, 472, 147-156.

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Figure 2. CVs of the CofC6-modified gold electrode at different scan speeds, 10, 20, 30, 40, 50 mV s-1 (smallest cycle to largest cycle), with Na2SO4 (0.2 M) as supporting electrolyte at pH 7. Only the reversible range (up to 50 mV s-1) is shown.

exclude that some flavin molecules are adsorbed nonspecifically by their imide substructure. With respect to our application it would be more favorable to cover the whole electrode surface by electrochemically inactive spacer units to avoid protein adsorption and, at the same time, to have some highly flexible flavin derivatives between to enable ET. It has been pointed out that mixtures of two different alkanethiols, having similar chain lengths, form completely miscible binary monolayers,28 whereas different chain lengths form preferentially distinctive domains.29 Taking these observations into account, we followed the surface modification strategy applied by Zimmermann et al.30 with small changes. Figure 1 shows the immobilization procedure employed in this work: In the first step a mixture of 3,3′-dithiodipropionic acid and its activated N-succinimidyl ester is adsorbed whereupon the ratio between activated and nonactivated carboxylic acid is variable. In the next step we link a synthetic flavin cofactor via an amide bond. The synthesis of the modified flavin cofactor, denoted CofC6, because it bears a hexyl (C6) amino group at N(10), was carried out almost analogously to the synthesis of a homologue ethyl amino derivative.31 Electrochemistry of a CofC6-Modified Electrode. Figure 2 shows the CVs of CofC6 immobilized on a gold electrode as described above, at different scan speeds (10-50 mV s-1) with Na2SO4 (0.2 M) as supporting electrolyte at pH 7. One reductive wave is detected at -465 mV vs SCE (-225 mV vs NHE). The observed reductive wave is assigned to a two-electron, oneproton reduction. The first reduction of the flavin is followed by protonation, and then the second reduction takes place immediately because the redox potential for the second reduction is more positive than for the first one. This sequence of electrochemical step-chemical step-electrochemical step (ece mechanism) is known for many flavin derivatives.17,32 Depending on pH, the resulting anionic flavohydroquinone might be further protonated (second chemical step) resulting in an ecec mechanism. For a reversible redox process the peak separation between cathodic and anodic current should be independent of scan speed, and at finite diffusion the current should increase linearly with scan speed. These conditions are only fulfilled at low scan speeds, as it can be seen by the CVs in Figure 2 and by the graphs in (28) Arnold, S.; Feng, Z. Q.; Kakiuchi, T.; Knoll, W.; Niki, K. J. Electroanal. Chem. 1997, 438, 91-97. (29) Hobara, D.; Ota, M.; Imabayashi, S.-i.; Niki, K.; Kakiuchi, T. J. Electroanal. Chem. 1998, 444, 113-119. (30) Zimmermann, H.; Lindgren, A.; Schuhmann, W.; Gorton, L. Chem. Eur. J. 2000, 6, 592-599. (31) Butenandt, J.; Epple, R.; Wallenborn, E.-U.; Eker, A. P. M.; Gramlich, V.; Carell, T. Chem. Eur. J. 2000, 6, 62-72. (32) Janik, B.; Elving, P. J. Chem. ReV. 1968, 68, 295-319.

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Figure 3. The relation of peak separation between cathodic and anodic current and scan speed is depicted on the left, and the increase of current with scan speed, on the right. At low scan speed, up to 50 mV s-1, the peak separation (potential difference ∆Ep between maximum cathodic and anodic current) stays constant and is about 55 mV. At faster scan speed (we measured up to 4000 mV s-1), the peak separation increases with scan speed as it is expected for quasi-reversible ET. The CVs in Figure 2 confirm this behavior, since the increase of cathodic and anodic current with scan speed is linear up to 50 mV s-1. At faster scan speed the slope of the corresponding curves decreases remarkably, as shown in Figure 3. To investigate the dependency of the redox potential Eq/hq (flavoquinone/flavohydroquinone) on pH, we carried out cyclic voltammetry at different pH. The resulting CVs and a graph showing dependency of the redox potential Eq/hq on the pH are shown in Figure 4. After changing the pH it was necessary to wait for 20-30 min until the redox potential equilibrated to a constant value. The modified electrode surface was found to be stable for hours at various pH values. These results confirm our assumption that CofC6 is linked at the electrode surface by a covalent amide bond and not just by electrostatic interaction. As pointed out in the literature33 and references given therein, the formal redox potentials of many flavins (in solution as well as adsorbed on various electrode materials) are in the range of -460 up to -480 mV vs SCE at pH 7. The redox potential of CofC6 (-465 mV vs SCE) is perfectly in line with this observation. Those authors also report that in the region of pH 3-8 the formal redox potentials of adsorbed flavins shift with a slope of about -50 - -60 mV per pH unit, whereas, in more basic media, a weaker pH dependence is observed. As shown in Figure 4, the pH dependence of a CofC6-modified electrode was found to be almost in this range. A value of -49 mV per pH unit in the region of pH 5-8 was determined by least-squares fit. This is somewhat lower than the theoretically expected value of -59 mV per pH unit according to Nernst for the overall reaction Fl + 2e- + 2H+ ) FlH2, whereas a pH dependency of -30 mV per pH unit is expected for the reaction Fl + 2e- + H+ ) FlH-. The pKa of flavohydroquinone9 in solution (that may differ from adsorbed flavohydroquinone) is about 6.2, indicating that at values below pH 6.2 the pH dependence of the redox potential should be -59 mV per pH unit and -30 mV per pH unit at high pH. At higher pH (>pH 8), the pH dependence of the CofC6-modified electrode decreases remarkably. In contrast to our prior assumption these observations indicate that the reduction of CofC6 adsorbed at the electrode surface is a two-electron, two-proton reduction at pH 7 (following an ecec mechanism), whereas the reduction of free flavins in solution is a two-electron, one-proton reduction at pH 7. This is also found for free CofC6 (see Supporting Information). A shift of the pKa to higher values upon absorption has been reported previously for other flavins.33,34 For an efficient catalysis of the protein ET by a hopping mechanism (electrode f flavin f flavoprotein) the ET rate from the electrode to the attached flavin mediator CofC6 is a crucial parameter. We estimated the ET rate constant by applying the Laviron approach:21 The CV of a diffusion-inhibited and reversible one-electron redox reaction shows no peak separation. Upon increasing the scan speed V, an increasing peak separation appears. ET becomes quasireversible or even irreversible.21,35,36 (33) Garjonyte, R.; Malinauskas, A.; Gorton, L. Bioelectrochemistry 2003, 61, 39-49. (34) Mallik, B.; Gani, D. J. Electroanal. Chem. 1992, 326, 37-49. (35) Armstrong, F. A.; Camba, R.; Heering, H. A.; Hirst, J.; Jeuken, L. J. C.; Jones, A. K.; Leger, C.; McEvoy, J. P. Faraday Discuss. 2000, 116, 191-203.

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Figure 3. Peak separation vs scan speed (left) and cathodic and anodic current vs scan speed (right) for CofC6 (adsorbed on a modified gold electrode) at various scan speeds.

Figure 4. CVs of CofC6 at different pHs, with scan speed V ) 50 mV s-1 (left), and a plot showing the dependence of Eq/hq on the pH (right). All CVs have been measured with the same electrode. Measurements have been started at pH 7 going first to higher and afterward to lower pH values.

Figure 5. Plot of the cathodic and anodic peak potentials Epc and Epa vs log V at pH 7. The linear section is fitted in order to determine the ET rate constant by the Laviron approach (least-squares fit for all data with ∆EP > 200/n ) 100 mV; n ) number of transferred electrons).

As pointed out by Laviron21 for irreversible ET, a plot of the peak potentials Epc and Epa against the logarithm of scan speed yields two straight lines which allow the determination of the ET rate constant. Figure 5 shows a plot of the cathodic and anodic peak potentials with respect to the logarithm of the scan speed. The symmetric increase of Epc and Epa with log V indicates that the electron-transfer coefficient R ) 0.5. By a least-squares fit for all ∆Ep values greater than 100 mV, we determined the ET rate constant as k0 ) 5 s-1 at pH 7. (36) Chen, K.; Hirst, J.; Camba, R.; Bonagura, C. A.; Stout, C. D.; Burgess, B. K.; Armstrong, F. A. Nature 2000, 405 (6788), 814-817. Erratum Nature 2000, 407 (6800), 110.

We repeated our measurements at pH 5 and pH 12 (data not shown). In all cases we obtained values between 3 and 7 s-1 for k0, indicating that neither the first protonation-deprotonation step nor the second (at pH 5 and 7) is rate-determining in the ece(c) mechanism. The ET rate we obtained by attaching the isoalloxazine moiety with a relatively long and flexible saturated spacer is in the typical range of adsorbed flavins.27 ET rates depend strongly on the distance between flavin and electrode and on their relative orientation. For FMN adsorbed on a tin dioxide electrode an ET rate constant of 3.5 s-1 has been observed.19 If flavins are covalently linked at the electrode, the ET rate is determined by the length and electronic nature of the spacer.37 In the case of an isoalloxazine derivative directly attached to a gold electrode by a thiopropyl chain (additionally stacking interactions could not be excluded) rate constants of 340 and 540 s-1 are reported for the cathodic and anodic process.27 Cyclic Voltammetry of the Flavoprotein WrbA Catalyzed by the CofC6-Modified Electrode. We first recorded CVs of the CofC6-modified electrode itself at different scan speeds (Figure 6, dotted lines). Thereafter, we added a solution of the flavoprotein WrbA and measured cyclic voltammetry under the same experimental conditions (0.2 M phosphate buffer, pH 7.2; supporting electrolyte, Na2SO4, 0.2 M). The concentration of WrbA was about 0.25 mM in FMN (448 nm ) 10.7 mM-1 cm-1; Hauska, unpublished). Figure 6 shows the resulting CVs at four different scan speeds (solid lines). The resulting current is the sum of the currents that belong to the reduction of CofC6 and WrbA. At 100 mV s-1, there is only a very low amount of (37) Liu, J.; Paddon-Row: M. N.; Gooding, J. J. J. Phys. Chem. B 2004, 108, 8460-8466.

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Figure 6. CVs of a CofC6-modified electrode before (dotted lines) and after addition of WrbA (solid lines) at different scan speeds (V) at pH 7.2 with Na2SO4 (0.2 M) as supporting electrolyte.

additional current belonging to the reduction of WrbA (compared to the dotted lines that are only caused by the reduction of CofC6). At 20 mV s-1, the current for protein reduction increases remarkably (the area below the cathodic and anodic peak is proportional to the amount of charge being transferred upon reduction and reoxidation). At 5 and 1 mV s-1, the current for protein reduction clearly dominates the amount of current that is necessary to charge the modified electrode surface. Usually, ET rates for proteins are very low. We conclude that the ET between electrode and WrbA can be regarded as a two-step hopping process38 (electrode f CofC6 f WrbA). If the ET is enabled via a two-stepsor even via a multistep hopping mechanismsthe slowest step will be rate-determining. As documented above, the reduction of the CofC6-modified electrode is only reversible up to a scan speed of 50 mV s-1, whereas at higher scan speed the reduction of CofC6 becomes kinetically hindered. The concentration of CofC6 at the electrode surface is low compared to the concentration of WrbA in the diffusion layer. Consequently, each CofC6 molecule has to be passed by several electrons on their way to WrbA. At 100 mV s-1 the first ET hopping step between electrode and CofC6 is too slow to enable the reduction of a significant amount of protein. Electrons, which arrived at CofC6, are immediately drawn back to the electrode before they could reach WrbA. When the scan speed is 100 times lower, a huge amount of protein is reduced. This reduction is catalyzed by the CofC6-modified electrode, where the first hopping step is the bottleneck in protein reduction, i.e., the rate-limiting step. With this electrode the redox potential of WrbA was determined at pH 7.2 as -490 mV vs Ag/AgCl (≈-250 mV vs NHE), as estimated from the peak maxima of cathodic and anodic current (bottom right panel in Figure 6). (38) Lambert, C.; No¨ll, G.; Schelter, J. Nat. Mater. 2002, 1, 69-73.

Because we had to use a miniaturized electrochemical cell for small sample volumes, we could not apply the more accurate SCE reference electrode. The redox potentials of WrbA and CofC6 immobilized at the electrode differ only marginally. This might be one reason for the ability of the modified electrode to catalyze protein ET on WrbA. To catalyze ET between electrode and WrbA, the formal redox potential of WrbA has to be in a range in which oxidized as well as reduced flavin is present at the electrode. The CVs of WrbA at the CofC6-modified electrode show peak separations of ∆E ) 70 mV at 100 mV s-1 and ∆E ) 50 mV for the scan speeds of 50 (data not shown), 20, 5, and 1 mV s-1. Hence, the overall ET is reversible at a scan speed of 50 mV s-1 and below, and the second hopping step, being responsible for the protein reduction, is fast compared to the first. A special interaction between flavin and WrbA, e.g. due to hydrogen bonding, could be responsible for the efficient ET. Such interaction may result in the formation of an active charge-transfer complex, in which the nature of ET would be intramolecular.39,40 Intramolecular ET, directed by a bridging subunit, is known to be faster than intermolecular ET. Consequently the flavin would act as a receptor unit that triggers ET by molecular recognition.41 At least the ET distance between flavin and the FMN cofactor inside WrbA has to be relatively short. Spectroelectrochemical Investigation of WrbA. The ability to catalyze protein ET by modification of a gold surface enabled us to investigate the spectroelectrochemical reduction of WrbA. A modified gold minigrid served as a transparent working electrode. The concentration of CofC6 adsorbed at the gold (39) Taube, H. Angew. Chem. 1984, 96, 315-326. (40) Taube, H. Science 1984, 226, 1028-1036. (41) Niemz, A.; Rotello, V. M. Acc. Chem. Res. 1999, 32, 44-52.

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Figure 7. Spectroelectrochemistry of WrbA at pH 7.2 at a concentration of about 0.25 mM (0.2 M phosphate buffer, 0.2 M Na2SO4). The reductive potential was increased in steps of 20 mV.

minigrid is too low to contribute to the absorption spectra. The experimental setup is described in the Supporting Information; a more detailed description is given elsewhere.26 Figure 7 shows the spectroelectrochemistry of WrbA at pH 7.2 at a concentration of about 0.25 mM. (0.2 M phosphate buffer, 0.2 M Na2SO4) with potential steps of 20 mV. Upon reduction of WrbA, the spectrum of the neutral oxidized protein changes to a spectrum that is characteristic for a flavoprotein in the fully reduced state.9,17,42 According to the literature42,43 the spectrum of neutral diprotonated flavohydroquinone of FMN shows a broad band at lowest energy with a maximum at 395 nm, whereas the anionic monoprotonated form has a maximum at 342 nm. With respect to these results the anionic monoprotonated flavohydroquinone of WrbA is obtained upon reduction. The isosbestic points indicate that, within the time scale of the experiment, only these two species are involved. After complete reduction, the potential was switched back to the oxidative region and the measured spectrum did not differ from the initial one, indicating complete reversibility of the observed process. If the protein would decompose (i.e. the protein denaturates or the cofactor is released), it would not be possible to obtain the initial spectrum. Even with smaller potential steps, no additional band was observed that would indicate the existence of a detectable neutral or anionic flavosemiquinone. In contrast to flaxodoxin, WrbA rapidly takes up two electrons, directly leading to the fully reduced form. This result indicates that, independent of structurally significant sequence similarity between flavodoxin and WrbA, the structure of the protein binding site must be significantly different in the two proteins. This conclusion is in agreement with the results of a previous biocomputing analysis.23

Conclusions By cyclic voltammetry and spectroelectrochemistry we showed that a flavin-modified gold electrode catalyzes the electrochemical (42) Ghisla, S.; Massey, V.; Lhoste, J. M.; Mayhew, S. G. Biochemistry 1974, 13, 589-597. (43) Shen, Z.; Prochazka, R.; Daub, J.; Fritz, N.; Acar, N.; Schneider, S. Phys. Chem. Chem. Phys. 2003, 5, 3257-3269.

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reduction of the flavoprotein WrbA. In contrast to flavodoxin,19 the reduction of WrbA occurs as two consecutive one-electron processes, without observing the semiquinone form as intermediate. Measurements by the equilibrium-redox-spectropotentiometry technique according to Dutton2 lead to the same finding (Hauska, unpublished). It is also worth mentioning that by cyclic voltammetry with an edge-oriented pyrolytic graphite (PG) working electrode we also found but one single quasireversible reductive wave for WrbA (data not shown). Flavodoxin and WrbA have quite different biological functions although similar biochemical activity, and this difference may be caused by their different ET properties. Flavodoxin is a typical ET protein that upon iron deficiencies replaces highly reductive ferredoxin in the assimilation of nitrogen and other processes.44 For these purposes, obviously an ET process with a semiquinone form as intermediate is employed. On the other hand, the putative function of WrbA is the catalysis of the obligatory two-electron reduction of quinones to hydroquinones. Relevant to this point is that intracellular reduction of quinones to semiquinones is deleterious, since semiquinones react with oxygen to give superoxide. Also other quinone oxidoreductases involved in oxidative-stress defense are known to use FAD as a prosthetic group and act by a two-electron reaction without accumulation of intermediate semiquinone.45,46 We are convinced that the ability of the flavin-modified electrode (-465 mV vs SCE at pH 7) to catalyze protein ET will also allow the investigation of further flavoproteins and even may be adopted for the investigation of redox proteins in general. In contrast to other methods applied in protein electrochemistry the protein stays in solution and no additional mediators are required. This setup could prove useful for UV/vis and also for FT-IR spectroelectrochemistry.47 To optimize the interaction between electrode and flavin, the length and structure of the spacer unit could be easily varied. Acknowledgment. This work was supported by the Deutsche Forschungsgemeinschaft DFG (GRK: 640 “Sensory Photoreceptors in Natural and Artificial Systems”). We thank Dr. Wolfgang Hackenbroch for helpful discussions and Dr. Bernhard Dick for kind support. Supporting Information Available: Information regarding the spectroelectrochemical setup, the protocol of the electrode surface modification procedure, synthesis of the flavin cofactor CofC6, the experimental part, UV/vis and fluorescence spectra of CofC6, and the spectroelectrochemistry of CofC6 (PDF). This material is available free of charge via the Internet at http://pubs.acs.org. LA051423N (44) Mayhew, S. G.; Ludwig, M. L. Enzymes, 3rd Ed.; Boyer, P. D., Ed.; Academic: New York, 1975; Vol. 12, pp 57-118. (45) Foster, C. E.; Bianchet, M. A.; Talalay, P.; Faig, M.; Amzel, L. M. Free Radical Biol. Med. 2000, 29, 241-245. (46) Chen, S.; Wu, K.; Knox, R. Free Radical Biol. Med. 2000, 29, 276-284. (47) Bueschel, M.; Stadler, C.; Lambert, C.; Beck, M.; Daub, J. J. Electroanal. Chem. 2000, 484, 24-32.