Electron transfer between the hydrogenase from Desulfovibrio vulgaris (Hildenborough) and viologens

Abstract

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...

The Covalent Linkage of a Viologen to a Flavoprotein Reductase Transforms it into an Oxidase

“…Cofactors are usually buried inside the polypeptide core of proteins in such a way that reaching them by using a non-specific substrate is quite unlikely. Viologens have been commonly used as mediators in enzymic reactions to improve the accessibility of prosthetic groups of enzymes to the substrate (Maeda and Kajiwara, 1985;Llobel et al, 1986;Adams et al, 1979) and also as mediators in electrochemical enzymic processes (Hoogvliet et al, 1988). In a previous study, we have shown that efficient electron exchange occurs between viologens and the flavoenzyme FNR when they are in solution (Bes et al, 1995).…”

Effect of the half wave potential and change of some viologens on the rate of conversion of crotonic acid into butyric acid by enoate reductase

“…The kinetic data listed in Table 1 are determined by means of chronoamperometry. The advantages of this technique over cyclic voltammetry and the assumptions made to calculate k' are discussed by Hoogvliet et al (1988aHoogvliet et al ( ,1988b. Table 1 shows the effect of the half wave potential and the net ionic charge on the pseudo first order rate constant of several viologens reacting with enoate reduetase.…”

“…Not much is known until now with respect to the kinetics and mechanism of interaction between viologens and the enzyme. As well as from the viewpoint of enzyme kinetics as for the development of a preparative bio-electrochemical reactor it is necessary to obtain information on the interaction between the mediator and the enzyme (Hoogvliet et al, 1988a;1988b). This paper describes the use of electrochemical methods (chronoamperometry and cyclic voltammetry) to obtain information on the conversion of crotonic acid into butyric acid by enoate reductase with a number of viologeus as the mediator and the consequences for the immobilization of the enzyme and the process conditicus to be chosen.…”

Voltammetric studies of the catalytic electron‐transfer process between the Desulfovibrio gigas hydrogenase and small proteins isolated from the same genus

“…From these voltammograms, the diffusion-controlled peak current, id.p, was determined. The same series of measurements was repeated af- For several reasons, however, some of the conditions could not be fulfilled (low enzyme and substrate concentrations, reversible instead of irreversible reactions and different diffusion coefficients for cytochrome c, and hydrogenase) [42]. Therefore, a typical set of A, versus llv [where v = potential scan rate (mVls)] plots yielded curved lines ( Fig.…”

“…It should be noted that the calculation of the rate constants uses a formalism that has a strong analogy with the use of the current (at t + 0) and enzyme activity measurepletion of substrate is avoided by measuring the initial velocity of the enzymic reaction [42]. …”