| Abstract
| - The nickel−iron hydrogenase from Chromatium vinosum adsorbs at a pyrolytic graphite edge-plane (PGE) electrode and catalyzes rapid interconversion of H+(aq) and H2 at potentials expected for thehalf-cell reaction 2H+ ⇄ H2, i.e., without the need for overpotentials. The voltammetry mirrorscharacteristics determined by conventional methods, while affording the capabilities for exquisite controland measurement of potential-dependent activities and substrate−product mass transport. Oxidation ofH2 is extremely rapid; at 10% partial pressure H2, mass transport control persists even at the highestelectrode rotation rates. The turnover number for H2 oxidation lies in the range of 1500−9000 s-1 at 30°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 controlled by processes occurring in the enzyme. Carbonmonoxide, which binds reversibly to the NiFe site in the active form, inhibits electrocatalysis and allowsimproved definition of signals that can be attributed to the reversible (non-turnover) oxidation and reductionof redox centers. One signal, at −30 mV vs SHE (pH 7.0, 30 °C), is assigned to the [3Fe-4S]+/0 clusteron the basis of potentiometric measurements. The second, at −301 mV and having a 1.5−2.5-fold greateramplitude, is tentatively assigned to the two [4Fe-4S]2+/+ clusters with similar reduction potentials. Noother redox couples are observed, suggesting that these two sets of centers are the only ones in CO-inhibited hydrogenase capable of undergoing simple rapid cycling of their redox states. With the buriedNiFe active site very unlikely to undergo direct electron exchange with the electrode, at least one andmore likely each of the three iron−sulfur clusters must serve as relay sites. The fact that H2 oxidation israpid even at potentials nearly 300 mV more negative than the reduction potential of the [3Fe-4S]+/0cluster shows that its singularly high equilibrium reduction potential does not compromise catalyticefficiency.
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