Enzyme electrokinetics: Energetics of succinate oxidation by fumarate reductase and succinate dehydrogenase

Protein film voltammetry is used to probe the energetics of electron transf er and substrate binding at the active site of a respiratory flavoenzyme-th e membrane-extrinsic catalytic domain of Escherichia coli fumarate reductas e (FrdAB). The activity as a function of the electrochemical driving force is revealed in catalytic voltammograms, the shapes of which are interpreted using a Michaelis-Menten model that incorporates the potential dimension. Voltammetric experiments carried out at room temperature under turnover con ditions reveal the reduction potentials of the FAD, the stability of the se miquinone, relevant protonation states, and pH-dependent succinate-enzyme b inding constants for all three redox states of the FAD. Fast-scan experimen ts in the presence of substrate confirm the value of the two-electron reduc tion potential of the FAD and show that product release is not rate limitin g. The sequence of binding and protonation events over the whole catalytic cycle is deduced. Importantly, comparisons are made with the electrocatalyt ic properties of SDH, the membrane-extrinsic catalytic domain of mitochondr ial complex II.