Direct Binding of Cysteine-367 Thiolate to the Active Site of the [FeFe]-Hydrogenase from Clostridium beijerinckii in the O ₂ -stable State

[FeFe]-hydrogenases are very active biocatalysts for H ₂ conversion. However, their active site is vulnerable to irreversible degradation initiated by O ₂ binding at the catalytic iron ion (Fe _d ) of the active center. CbA5H, the [FeFe]-hydrogenases from Clostridium beijerinckii exhibits stability towards oxygen (O ₂ ) due to its ability to reversibly enter an inactive state termed H _inact upon contact with O ₂ . We previously proposed that the close distance of approximately 3.1 Å between the thiol of a nearby cysteine (C367) and the Fe _d , based on a 2.9 Å crystal structure of CbA5H in the H _inact state, enables their binding to each other. This binding therefore was suggested to shield the Fe _d from O ₂ damage. However, there is currently a lack of evidence to support this hypothesis. Furthermore, density functional theory (DFT) calculations based on a homologous model favored hydroxide as the binding ligand of the Fe _d over the thiol of C367. In this study, we present the crystal structure of CbA5H in the H _inact state at an improved resolution of 2.15 Å. The structure reveals a direct binding between the thiol of C367 and the Fe _d with a distance of approximated 2.77 Å which is well supported by our DFT calculations based on the new crystallographic data. It is noteworthy that the 2.77 Å bond distance is strikingly long when compared with other iron-sulfur bonds. This finding may provide a crucial foundation for understanding the rapid reversibility of the H _inact state.