Spectroelectrochemistry of Redox Enzymes

Abstract: A low volume and long optical path spectroelectrochemical cell was constructed and tested in a number of applications such as detection of direct electron transfer (DET) between redox enzymes and gold electrodes, determination of the formal potentials of redox centres in proteins and enzymes, as well as mechanistic studies of heterogeneous and intermolecular electron transfer of copper oxidases and succinate:quinine oxidoreductase (Complex II). The existence of DET between enzymes and electrodes was demonstrated for theophylline oxidase, cytochrome P450 cam, laccase, bilirubin oxidase, sulphite oxidase and Complex II. Spectroelectrochemical investigations of the ET between blue multi-copper oxidases and gold electrodes demonstrated that the mechanism of heterogeneous ET of laccases on gold is completely different from that usually observed on carbon electrodes. At carbon electrodes laccases are electronically connected to the conducting electrode through the T1 copper centre. At gold surfaces, by contrast, the electronic connection of the laccase redox centre to the surface is established through the T2 copper centre. Such an ET connection between the gold surface and the T2 copper centre, severely disturbs the oxygen reduction reaction catalysed by laccase. Similar observation was made for two bilirubin oxidases. Spectroelectrochemical studies of Complex II in the gold capillary cell showed that DET for intact Complex II is realised through the heme center with a low redox potential, hemeL. The study of DET reactions in combination with specific ET mediators and inhibitors, suggested that a postulated proximal quinone binding site may indeed exist in the Complex II structure. Our spectroelectrochemical studies indicate that this quinone binding site seems to be closed when both high and low potential heme sites are reduced.