[NiFe] and [FeS] Cofactors in the Membrane-Bound Hydrogenase of Ralstonia eutropha Investigated by X-ray Absorption Spectroscopy: Insights into O2-Tolerant H2 Cleavage

Molecular features that allow certain [NiFe] hydrogenases to catalyze the conversion of molecular hydrogen (H2) in the presence of dioxygen (O2) were investigated. Using X-ray absorption spectroscopy (XAS), we compared the [NiFe] active site and FeS clusters in the O2-tolerant membrane-bound hydrogenase (MBH) of Ralstonia eutropha and the O2-sensitive periplasmic hydrogenase (PH) of Desulfovibrio gigas. Fe-XAS indicated an unusual complement of iron–sulfur centers in the MBH, likely based on a specific structure of the FeS cluster proximal to the active site. This cluster is a [4Fe4S] cubane in PH. For MBH, it comprises less than ~2.7 Å Fe–Fe distances and additional longer vectors of ≥3.4 Å, consistent with an Fe trimer with a more isolated Fe ion. Ni-XAS indicated a similar architecture of the [NiFe] site in MBH and PH, featuring Ni coordination by four thiolates of conserved cysteines, i.e., in the fully reduced state (Ni-SR). For oxidized states, short Ni−μO bonds due to Ni–Fe bridging oxygen species were detected in the Ni-B state of the MBH and in the Ni-A state of the PH. Furthermore, a bridging sulfenate (CysSO) is suggested for an inactive state (Niia-S) of the MBH. We propose that the O2 tolerance of the MBH is mainly based on a dedicated electron donation from a modified proximal FeS cluster to the active site, which may favor formation of the rapidly reactivated Ni-B state instead of the slowly reactivated Ni-A state. Thereby, the catalytic activity of the MBH is facilitated in the presence of both H2 and O2.