Electrochemical Definitions of O2Sensitivity and Oxidative Inactivation in Hydrogenases

Abstract

A new strategy is described for comparing, quantitatively, the ability of hydrogenases to tolerate exposure to O₂ and anoxic oxidizing conditions. Using protein film voltammetry, the inherent sensitivities to these challenges (thermodynamic potentials and rates of reactions) have been measured for enzymes from a range of mesophilic microorganisms. In the absence of O₂, all the hydrogenases undergo reversible inactivation at various potentials above that of the H⁺/H₂ redox couple, and H₂ oxidation activities are thus limited to characteristic “potential windows”. Reactions with O₂ vary greatly; the [FeFe]-hydrogenase from Desulfovibrio desulfuricans ATCC 7757, an anaerobe, is irreversibly damaged by O₂, surviving only if exposed to O₂ in the anaerobically oxidized state (which therefore affords protection). In contrast, the membrane-bound [NiFe]-hydrogenase from the aerobe, Ralstonia eutropha, reacts reversibly with O₂ even during turnover and continues to catalyze H₂ oxidation in the presence of O₂.