A novel electrochemical approach to the characterization of oxidoreductase reactions.

Abstract

Electrochemical methods based on enzyme-electrochemical reactions have been developed for studying oxidoreductase reactions. The methods measure a current resulting from an oxidoreductase reaction with an electrode serving as a final electron acceptor (or donor) in the reaction. A theoretical equation for the enzyme-electrochemical reaction, called bioelectrocatalysis, is derived, which enables kinetic analysis of the reaction. In combination with spectrophotometry, the electrochemical method provides a method for determining the redox potentials of proteins and enzymes. An alternative method based on bulk electrolysis in a quartz cell for UV-vis spectroscopy has been developed for the measurements of protein redox potentials on a conventional spectrophotometer. The electrochemical methods are applied to kinetic and thermodynamic analyses for the reactions of a variety of enzymes including a newly discovered enzyme, quinohemoprotein amine dehydrogenase (QH-AmDH), and bilirubin oxidase (BOD) [EC 1.3.3.5, from Myrothecium verrucaria], a copper-containing enzyme useful for bioelectrocatalytic O(2) reduction in biofuel cells. The electrochemical method for kinetic analysis has been successfully applied to the analysis of oxidoreductase reactions in vivo, as demonstrated by the reaction of glucose dehydrogenase in Escherichia coli. The advantages of the electrochemical methods are discussed.