Hydrogen and oxygen trapping at the H-cluster of [FeFe]-hydrogenase revealed by site-selective spectroscopy and QM/MM calculations

[FeFe]-hydrogenases are superior hydrogen conversion catalysts. They bind a cofactor (H-cluster) comprising a four-iron and a diiron unit with three carbon monoxide (CO) and two cyanide (CN-) ligands. Hydrogen (H-2) and oxygen (O-2) binding at the H-cluster was studied in the C169A variant of [FeFe]-hydrogenase HYDA1, in comparison to the active oxidized (Hox) and CO-inhibited (Hox-CO) species in wildtype enzyme. Fe-57 labeling of the diiron site was achieved by in vitro maturation with a synthetic cofactor analogue. Site-selective X-ray absorption, emission, and nuclear inelastic/forward scattering methods and infrared spectroscopy were combined with quantum chemical calculations to determine the molecular and electronic structure and vibrational dynamics of detected cofactor species. Hox reveals an apical vacancy at Fe-d in a [4Fe4S-2Fe](3-) complex with the net spin on Fe-d whereas Hox-CO shows an apical CN- at Fe-d in a [4Fe4S-2Fe(CO)](3-) complex with net spin sharing among Fe-p and Fe-d (proximal or distal iron ions in [2Fe]). At ambient O-2 pressure, a novel H-cluster species (Hox-O-2) accumulated in C169A, assigned to a [4Fe4S-2Fe(O-2)](3-) complex with an apical superoxide (O-2(-)) carrying the net spin bound at Fe-d. H-2 exposure populated the two-electron reduced Hhyd species in C169A, assigned as a [(H)4Fe4S-2Fe(H)](3-) complex with the net spin on the reduced cubane, an apical hydride at Fe-d, and a proton at a cysteine ligand. Hox-O-2 and Hhyd are stabilized by impaired O-2(-) protonation or proton release after H-2 cleavage due to interruption of the proton path towards and out of the active site.