Caught in the H : Crystal Structure and Spectroscopy Reveal a Sulfur Bound to the Active Site of an O -stable State of [FeFe] Hydrogenase.

[FeFe] hydrogenases are the most active H 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 -stable state called H . To date, the structure and mechanism of formation of H 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 state and its mechanism of formation. This may facilitate the design of O -stable hydrogenases and molecular catalysts.