The metabolism of many anaerobes relies on [NiFe]-hydrogenases, whose characterization when bound to substrates has proven non-trivial. Presented here is direct evidence for a hydride bridge in the active site of the ⁵⁷Fe-labelled fully reduced Ni-R form of Desulfovibrio vulgaris Miyazaki F [NiFe]-hydrogenase. A unique ‘wagging’ mode involving H⁻ motion perpendicular to the Ni(μ-H)⁵⁷Fe plane was studied using ⁵⁷Fe-specific nuclear resonance vibrational spectroscopy and density functional theory (DFT) calculations. On Ni(μ-D)⁵⁷Fe deuteride substitution, this wagging causes a characteristic perturbation of Fe–CO/CN bands. Spectra have been interpreted by comparison with Ni(μ-H/D)⁵⁷Fe enzyme mimics [(dppe)Ni(μ-pdt)(μ-H/D)⁵⁷Fe(CO)₃]⁺ and DFT calculations, which collectively indicate a low-spin Ni(II)(μ-H)Fe(II) core for Ni-R, with H⁻ binding Ni more tightly than Fe. The present methodology is also relevant to characterizing Fe–H moieties in other important natural and synthetic catalysts.

[FeFe] hydrogenases are extremely active H₂ converting enzymes. Their mechanism remains highly controversial, in particular, the nature of the one-electron and two-electron reduced intermediates called H_redH⁺ and H_sredH⁺. In one model, the H_redH⁺ and H_sredH⁺ states contain a semi-bridging CO, while in the other model, the bridging CO is replaced by a bridging hydride. Using low temperature infra-red (IR) spectroscopy and nuclear resonance vibrational spectroscopy (NRVS), together with density functional theory (DFT) calculations, we show that the bridging CO is retained in the H_sredH⁺ and H_redH⁺ states in the [FeFe] hydrogenases from Chlamydomonas reihnhardtii (CrHydA1) and Desulfovibrio desulfuricans (DdHydAB), respectively. Furthermore, there is no evidence for a bridging hydride in either state. These results agree with a model of the catalytic cycle in which the H_redH⁺ and H_sredH⁺ states are integral, catalytically competent components. We conclude that proton-coupled electron transfer between the two subclusters is crucial to catalysis and allows these enzymes to operate in a highly efficient and reversible manner.

[FeFe] hydrogenases are the most active H₂ converting catalysts in nature, but their extreme oxygen sensitivity limits their use in technological applications. The [FeFe] hydrogenases from sulfate reducing bacteria can be purified in an O₂‐stable state called H_inact. To date, the structure and mechanism of formation of H_inact remain unknown. Our 1.65 Å crystal structure of this state reveals a sulfur ligand bound to the open coordination site. Furthermore, in‐depth spectroscopic characterization by X‐ray absorption spectroscopy (XAS), nuclear resonance vibrational spectroscopy (NRVS), resonance Raman (RR) spectroscopy and infrared (IR) spectroscopy, together with hybrid quantum mechanical and molecular mechanical (QM/MM) calculations, provide detailed chemical insight into the H_inact state and its mechanism of formation. This may facilitate the design of O₂‐stable hydrogenases and molecular catalysts.