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FeFe hydrogenases are the most efficient H2-producing enzymes. However, inactivation by O2remains an obstacle that prevents them being used in many biotechnological devices. Here, we combine electrochemistry, site-directed mutagenesis, molecular dynamics and quantum chemical calculations to uncover the molecular mechanism of O2diffusion within the enzyme and its reactions at the active site. We propose that the partial reversibility of the reaction with O2results from the four-electron reduction of O2to water. The third electron/proton transfer step is the bottleneck for water production, competing with formation of a highly reactive OH radical and hydroxylated cysteine. The rapid delivery of electrons and protons to the active site is therefore crucial to prevent the accumulation of these aggressive species during prolonged O2exposure. These findings should provide important clues for the design of hydrogenase mutants with increased resistance to oxidative damage.