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Hydrogenases are efficient biological catalysts of H₂ oxidation and production. Most of them are inhibited by O₂, and a prerequisite for their use in biotechnological applications under air is to improve their oxygen tolerance. We have previously shown that exchanging the residue at position 74 in the large subunit of the oxygen-sensitive [NiFe] hydrogenase from Desulfovibrio fructosovorans could impact the reaction of the enzyme with O₂ (Dementin, S.; J. Am. Chem. Soc. 2009, 131, 10156−10164; Liebgott, P. P.; Nat. Chem. Biol. 2010, 6, 63−70). This residue, a valine in the wild-type enzyme, located at the bottleneck of the gas channel near the active site, has here been exchanged with a cysteine. A thorough characterization using a combination of kinetic, spectroscopic (EPR, FTIR), and electrochemical studies demonstrates that the V74C mutant has features of the naturally occurring oxygen-tolerant membrane …