Direct Bindingof Cysteine-367 Thiolate to the ActiveSite of the [FeFe]-Hydrogenase from in the O-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]-hydrogenase from , exhibits stability toward 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 & Aring; between the thiol of a nearby cysteine (C367) and Fe-d, based on a 2.9 & Aring; crystal structure of CbA5H in the H-inact state, enables their binding to each other. This binding therefore was suggested to shield 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 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 & Aring;. The structure reveals a direct binding between the thiol of C367 and Fe-d with a distance of approximately 2.77 & Aring;, which is well supported by our DFT calculations based on the new crystallographic data. It is noteworthy that the 2.77 & Aring; 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.