Catalytic electron transport in Chromatium vinosum [NiFe]-hydrogenase: Application of voltammetry in detecting redox-active centers and establishing that hydrogen oxidation is very fast even at potentials close to the reversible H+/H-2 value

The nickel-iron hydrogenase from Chromatium vinosum adsorbs at a pyrolytic graphite edge-plane (PGE) electrode and catalyzes rapid interconversion of H-(aq)(+) and H-2 at potentials expected for the half-cell reaction 2H(+) r eversible arrow H-2, i.e., without the need for overpotentials. The voltamm etry mirrors characteristics determined by conventional methods, while affo rding the capabilities for exquisite control and measurement of potential-d ependent activities and Substrate-product mass transport. Oxidation of H-2 is extremely rapid; at 10% partial pressure H-2, mass transport control per sists even at the highest electrode rotation rates. The turnover number for H-2 oxidation lies in the range of 1500-9000 s(-1) at 30 degrees C (pH 5-8 ), which is significantly higher than that observed using methylene blue as the electron acceptor. By contrast, proton reduction is slower and control led by processes occurring in the enzyme-Carbon monoxide, which binds rever sibly to the NiFe site in the active form, inhibits electrocatalysis and al lows improved definition of signals that can be attributed to the reversibl e (non-turnover) oxidation and reduction of redox centers. One signal, at - 30 mV vs SHE (pH 7.0, 30 degrees C), is assigned to the [3Fe-4S](+/0) clust er on the basis of potentiometric measurements. The second, at -301 mV and having a 1.5-2.5-fold greater amplitude, is tentatively assigned to the two [4Fe-4S]2(+/+) clusters with similar reduction potentials. No other redox couples are observed, suggesting that these two sets of centers are the onl y ones in CO-inhibited hydrogenase capable of undergoing simple rapid cycli ng of their redox states. With the buried NiFe active site very unlikely to undergo direct electron exchange with the electrode, at least one and more likely each of the three iron-sulfur clusters must serve as relay sites. T he fact that H-2 oxidation is rapid even at potentials nearly 300 mV more n egative than the reduction potential of the [3Fe-4S](+/0) cluster shows tha t its singularly high equilibrium reduction potential does not compromise c atalytic efficiency.