A safety cap protects hydrogenase from oxygen attack

[FeFe]-hydrogenases are efficient H2-catalysts, yet upon contact with dioxygen their catalytic cofactor (H-cluster) is irreversibly inactivated. Here, we combine X-ray crystallography, rational protein design, direct electrochemistry, and Fourier-transform infrared spectroscopy to describe a protein morphing mechanism that controls the reversible transition between the catalytic Hox-state and the inactive but oxygen-resistant Hinact-state in [FeFe]-hydrogenase CbA5H of Clostridium beijerinckii. The X-ray structure of air-exposed CbA5H reveals that a conserved cysteine residue in the local environment of the active site (H-cluster) directly coordinates the substrate-binding site, providing a safety cap that prevents O2-binding and consequently, cofactor degradation. This protection mechanism depends on three non-conserved amino acids situated approximately 13 A away from the H-cluster, demonstrating that the 1st coordination sphere chemistry of the H-cluster can be remote-controlled by distant residues. [FeFe]-hydrogenases catalyze the conversion of protons and electrons to molecular hydrogen, but upon exposure to oxygen, their catalytic cofactor is irreversibly inactivated. Here, the authors determine the crystal structure of hydrogenase CbA5H and identify a cysteine residue, which acts as a safety cap that shields the active site from oxygen.