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The electron transfer kinetics between the hydrogenase from Desulvovibrio vulgaris (strain Hildenborough) and three different viologen mediators has been investigated by cyclic voltammetry. The mediators methyl viologen, di(n-aminopropyl) viologen and propyl viologen sulfonate differ in redox potential and in net charge. Dependent on the pH both the one-and two-electron-reduced forms or only the two-electron-reduced form of the viologens are effective in electron exchange with hydrogenase. Calculations of the second-order rate constant k for the reaction between reduced viologen and hydrogenase are based on the theory of the simplest electrocatalytic mechanism. Values for k are in the range of 106-107 M-' s-' and increase in the direction propyl viologen sulfonate + methyl viologen + di(n-aminopropyl) viologen. An explanation is based on electrostatic interactions. It is proposed that the electron transfer reaction is the rate-determining step in the catalytic mechanism.In recent years the study of electron-transfer processes in biological systems by means of electrochemical methods has received increasing attention. For a number of small-sized redox proteins (e. g. cytochrome c, ferredoxines, and azurine) a direct electron transfer at metal oxide electrodes [l] or at gold electrodes modified with organic non-electroactive organic 'promotors' [2] has been achieved. The electrode kinetics of these systems can be investigated by standard techniques like cyclic voltammetry, rotating-disk electrode or impedance measurements. Still, for most (larger-sized) redox enzymes no direct electron transfer at electrodes has been described yet. The literature on the direct electron transfer between redox enzymes and electrodes is still scarce. Some examples are hydrogenase at the dropping mercury electrode in the presence of polylysine [3], covalently modified glucose oxidase at metal Correspandence to J. C. Hoogvliet,

The electron transfer kinetics between the hydrogenase from Desuljovibrio vulgaris (strain Hildenborough) and the mediators methyl viologen, di-(n-aminopropyl) viologen and propyl viologen sulfonate have been investigated by chronoamperometry. Second-order rate constants were calculated on basis of the theory for a simple catalytic mechanism and are compared with the results obtained before by cyclic voltammetry (preceding paper in this journal). From the ionic-strength dependence and the observed differences in the rate constants for the differently charged viologens, the existence of an electrostatic interaction between mediator and a negatively charged part of the protein is confirmed. Chronoamperometry (computer-controlled) was found to possess advantages over cyclic voltammetry in the determination of homogeneous rate constants (faster, more accurate, and better reproducibility).In the preceding paper [l] we described the results of electrochemical investigations of electron-transfer characteristics for the reaction between the hydrogenase from Desulfovibrio vulgaris (strain Hildenborough) and three differently charged viologen mediators, using the technique of cyclic voltammetry.Although cyclic voltammetry proved to be a valuable technique for kinetic studies of electron transfer reactions of redox enzymes [2 -51, there are also some disadvantages, especially in those cases where the reaction mechanism cannot be described by the theory of the simple electrocatalytic mechanism [6,7] (pseudo-first-order and irreversible chemical reactions) without the use of important simplifications [l, 81. The simplifications which have been made to apply the theory of a simple catalytic mechanism to more complicated reactions (second-order kinetics, reversible chemical reactions) imply the extrapolation of measured data towards infinite scan rate. The main disadvantage of this approximation is that cyclic n, number of electrons involved in the electrode reaction; Q, net charge of the fully reduced form (V") of the mediator; t , time (s); 2, kinetic parameter, defined as /z = k c; t (dimensionless); erf(x), error function defined as erf(x) = -J"e-y2 dy. "x '12 0 voltammetry cannot be used for accurate measurements at high scan rates (> 200 mV/s) with solid electrodes, unless impractically high concentrations are employed. A second disadvantage is that quite a large number of measurements is required at a number of scan rates and enzyme concentrations to complete a kinetic study.In this paper we report results on chronoamperometric kinetic studies of the hydrogenase reduction mediated by viologens. The major object of the present study was to investigate whether the use of this technique would enable us to overcome the above-mentioned limitations of the cyclic voltammetric studies in the case of a complicated reaction mechanism.The application of chronoamperometry for kinetic studies of redox proteins has been described before. Ryan et al. compared chronoamperometry with stopped-flow spectrophotometry for the determination...