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Hydrogenases are metalloenzymes that catalyze the reversible oxidation of molecular hydrogen into protons and electrons. For this purpose, [FeFe]-hydrogenases utilize a hexanuclear iron cofactor, the H-cluster. This biologically unique cofactor provides the enzyme with outstanding catalytic activities, but it is also highly oxygen sensitive. Under in vitro conditions, oxygen stable forms of the H-cluster denoted H_trans and H_inact can be generated via treatment with sulfide under oxidizing conditions. Herein, we show that an H_trans-like species forms spontaneously under intracellular conditions on a time scale of hours, concurrent with the cells ceasing H₂ production. Addition of cysteine or sulfide during the maturation promotes the formation of this H-cluster state. Moreover, it is found that formation of the observed H_trans-like species is influenced by both steric factors and proton transfer, underscoring the importance …