The electron-transfer kinetics between three different mediators and the hexahemic enzyme nitrite reductase isolated from Desulfovibrio desulfuricans (ATCC 27774) were investigated by cyclic voltammetry and by chronoamperometry. The mediators, methyl viologen, Desulfovibrio vulgaris (Hildenborough) cytochrome c3 and D. desulfiricans (ATCC 27774) cytochrome c3 differ in structure, redox potential and charge. The reduced form of each mediator exchanged electrons with nitrite reductase. Second-order rate constants, k, were calculated on the basis of the theory for a simple catalytic mechanism and the results, obtained by cyclic voltammetry, were compared with those obtained by chronoamperometry. Values for k are in the range lo6-lo8 M-' s-' and increase in the direction D. desulfiricans cytochrome c3 -+ D. vulgaris cytochrome c3 + methyl viologen.An explanation is advanced on the basis of electrostatic interactions and relative orientation between the partners involved.Chronoamperometry (computer controlled) offers advantages over cyclic voltammetry in the determination of homogeneous rate constants (faster, more accurate and better reproducibility).Direct, unmediated electrochemical responses of the hexaheme nitrite reductase were also reported.The study of electron-transfer processes in biological systems by electrochemical methods has recently received increasing attention. For a number of a small-sized redox proteins (e.g. cytochrome c, cytochrome c3 and ferredoxins) a direct electron transfer at metal oxide electrodes [l] or at carbon electrodes [2,3] has been achieved. The electrode kinetics of these systems can be investigated by such standard techniques as cyclic voltammetry, rotating-disk electrode or impedance measurements. Still, for larger-sized redox enzymes, no direct electron transfer at electrodes has yet been described. Reports of direct electron transfer between redox enzymes and electrodes remain scarce. Some examples are studies of hydrogenase at the dropping mercury electrode in the presence of polylysine [4], covalently modified glucose oxidase at metal electrodes [5] and cytochrome c peroxidase at tin oxide electrodes [6].Coupling an enzyme to an electrochemical reaction by use of redox mediators offers an alternative approach. Voltammetric and chronoamperometric methods can be used to measure the homogeneous electron transfer rates between redox enzymes and artificial mediators or between redox en-
The kinetics of electron transfer between the Desulfovibrio gigas hydrogenase and several electron-transfer proteins from Desulfovibrio species were investigated by cyclic voltammetry, squarewave voltammetry and chronoamperometry. The cytochrome c3 from Desulfovibrio vulgaris (Hildenborough), Desulfovibrio desulfuricans (Norway 4), Desu@vibrio desulfuricans (American Type Culture Collection 27774) and D. gigas (NCIB 9332) were used as redox carriers. They differ in their redox potentials and isoelectric point. Depending on the pH, all the reduced forms of these cytochromes were effective in electron exchange with hydrogenase. Other small electron-transfer proteins such as ferredoxin I, ferredoxin I1 and rubredoxin from D. gigas were tentatively used as redox carriers. Only ferredoxin I1 was effective in mediating electron exchange between hydrogenase and the working electrode. The second-order rate constants k for the reaction between reduced proteins and hydrogenase were calculated based on the theory of the simplest electrocatalytic mecha- Hydrogenase catalyzes the simple and reversible reaction H, * 2H' + 2e in the presence of suitable electron donors/ acceptors. This enzyme, present in sulfate-reducing bacteria, plays a central role in hydrogen metabolism [l]. A number of hydrogenases have been obtained from various sources, exhibiting different properties with respect to specific activity, oxygen stability, activation behavior, metal content, subunit structure and electron-carrier specificity [ 1 -51.The hydrogenase from the sulfate-reducing anaerobic bacterium Desulfovibrio gigas (molecular mass of 89 kDa with subunits of 26 kDa and 63 kDa [6, 71) contains four redox centers; one nickel center, one [3Fe-4S] and two [4Fe-4S] clusters, whose existence was proven by electron paramagnetic resonance and Mossbauer spectroscopic studies [7,
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