Caught in the H(inact): Crystal Structure and Spectroscopy Reveal a Sulfur Bound to the Active Site of an O(2)‐stable State of [FeFe] Hydrogenase

[FeFe] hydrogenases are the most active H(2) 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 O(2)‐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 O(2)‐stable hydrogenases and molecular catalysts.