Fast-scan cyclic voltammetry of protein films on pyrolytic graphite edge electrodes: characteristics of electron exchange

The rapid electron-exchange characteristics of metalloproteins adsorbed at a pyrolytic graphite "edge" electrode have been studied by analog dc cyclic voltammetry at scan rates up to 3000 V s-1. The voltammetry of four proteins, azurin (a "blue" copper protein) and three 7Fe ferredoxins, reveals oxidation and reduction peaks that display only modest increases in width and peak separation as the scan rate is raised. This is indicative of a substantially homogeneous population of noninteracting centers which undergo rapid electron exchange with the electrode. Both the Butler--Volmer and Marcus models have been tested. The electrochemical kinetics, as reflected by k0 (the rate at zero overpotential), are too fast to allow the determination of reorganization energies by this method. Nonetheless, the rapid and energetically coherent nature of the electron transfer enables the cyclic oxidation and reduction of protein redox centers to be examined on a time scale sufficiently short to recognize coupled processes occurring in the millisecond time domain, which are characteristic of the protein under investigation. Two of the ferredoxins display increasingly asymmetric voltammetry as the scan rate is increased, which is attributed to the coupling of electron transfer to conformational (or orientational) changes. For azurin, the use of higher electrolyte concentrations enables studies to be made at scan rates up to 3000 V s-1, from which a standard electron-transfer rate constant in the region of 5000 s-1 is obtained. At these high scan rates, azurin still shows very symmetrical voltammograms but with peak shapes displaying a more gradual decrease in current, at increasing overpotential, than is predicted using realistic values of the reorganization energy. The ability to measure even faster rate constants and access coupled reactions occurring in shorter time domains is likely to be limited by complex processes occurring on the graphite surface.