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Hydrogenases are abundant enzymes that catalyse the reversible interconversion of H₂ into protons and electrons at high rates. Those hydrogenases maintaining their activity in the presence of O₂ are considered to be central to H₂-based technologies, such as enzymatic fuel cells and for light-driven H₂ production. Despite comprehensive genetic, biochemical, electrochemical and spectroscopic investigations^,,,,,, the molecular background allowing a structural interpretation of how the catalytic centre is protected from irreversible inactivation by O₂ has remained unclear. Here we present the crystal structure of an O₂-tolerant [NiFe]-hydrogenase from the aerobic H₂ oxidizer Ralstonia eutropha H16 at 1.5 Å resolution. The heterodimeric enzyme consists of a large subunit harbouring the catalytic centre in the H₂-reduced state and a small subunit containing an electron relay consisting of three different iron-sulphur …