Artur Sucheta

Voltammetric studies of redox-active centers in metalloproteins adsorbed on electrodes

Publisher Summary In recent years, redox proteins can be induced to interact directly with an electrode surface and display reversible electrochemistry in the same way as many smaller molecules. This has suggested the possibility of using dynamic electrochemical methods, such as cyclic voltammetry, to examine the many intricate functional properties of redox-active centers in proteins. This chapter describes a particular strategy, thus far demonstrated to be applicable for investigating labile Fe-S clusters, which could be more widely exploited for deciphering complicated reactivity that is linked to electron transfer. The discussion here focuses on application. It first outlines some general features of voltammetric methods that are potentially useful for studies of redox sites in proteins. Although considerable effort may be required to establish conditions for obtaining a stable, electroactive film, the voltammetric approach can lead to the detection and clarification of chemistry that is not revealed by other methods. A wide spectrum of information on dynamic systems can be derived, ranging from a rapid image of the redox chemistry of centers in a protein to the determination of equilibrium and kinetic constants for coupled reactions. It permits an extensive exploration of reactivities with small amounts of material and is useful in the characterization of labile systems for which critical conditions must be met for preparation of spectroscopic samples.

Classification of fumarate reductases and succinate dehydrogenases based upon their contrasting behaviour in the reduced benzylviologen/fumarate assay

Reduction of fumarate by soluble beef heart succinate dehydrogenase has been shown previously by voltammetry to become increasingly retarded as the potential is lowered below a threshold potential of −80 mV at pH 7.5. The behaviour resembles that of a tunnel diode, an electronic device exhibiting the property of negative resistance. The enzyme thus acts to oppose fumarate reduction under conditions of high thermodynamic driving force. We now provide independent evidence for this phenomenon from spectrophotometric kinetic assays. With reduced benzylviologen as electron donor, we have studied the reduction of fumarate catalysed by various enzymes classified either as succinate dehydrogenases or fumarate reductases. For succinate dehydrogenases, the rate increases as the concentration of reduced dye (driving force) decreases during the reaction. In contrast, authentic fumarate reductases of anaerobic cells (and ‘succinate dehydrogenase’ from Bacillus subtilis) neither exhibit the electrochemical effect nor deviate from simple kinetic behaviour in the cuvette assay. The ‘tunnel‐diode’ effect may thus represent an evolutionary adaptation to aerobic metabolism.