Stability of the H-cluster under whole-cell conditions—formation of an H(trans)-like state and its reactivity towards oxygen

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 H(2) 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 of outer coordination sphere effects on H-cluster reactivity. GRAPHICAL ABSTRACT: [Image: see text] SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s00775-022-01928-5.