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

Abstract

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.

Resonance Raman Spectroscopic Analysis of the [NiFe] Active Site and the Proximal [4Fe-3S] Cluster of an O2-Tolerant Membrane-Bound Hydrogenase in the Crystalline State

“…6 The minor contribution originates from an electron paramagnetic resonance-silent Ni(II) species, which has been already observed in previous studies on the MBH. 9,18 According to previous suggestions, 21 it might be a "ready" species (Ni r -S) with either a hydroxyl or a water molecule in the bridging position. An alternative assignment might be a structure with an open coordination site between Fe(II) and Ni(II), designated as "active" Ni a -S state ( Figure 1).…”

Bacterial formate hydrogenlyase complex

“…Metal analysis by ICP-MS was performed as a service by the School of Chemistry, University of Edinburgh. TXRF analysis was carried out on a PicoFox spectrometer (Bruker) as previously described (49,50). A gallium elemental concentration standard (Sigma) was added to the protein solutions (1:1 vol/vol) before the measurements.…”

Multiscale Simulations Give Insight into the Hydrogen In and Out Pathways of [NiFe]-Hydrogenases from Aquifex aeolicus and Desulfovibrio fructosovorans

“…Although several results have been obtained through mutations, [11][12][13]68,79 this hypothesis has been superseded by the resolution of several oxygen resistant Hases. [14][15][16][17][19][20][21][22]81 To check the influence of the tunnel size, the total volume of the tunnels ( Figure SI5) as well as the distance V64 L −L112 L between the Cβ of L122 and Cγ of V64 have been measured ( Figure SI6). The distribution of the sections is centered at 1 nm for AaHase and 0.9 nm for DfHase (in line with the average value of 0.8 nm found in ref 84), in agreement with previous data showing that this Val-Leu gate diameter is larger in the O 2 -tolerant MBH from R. eutropha than in the O 2 -sensitive hydrogenases from D. vulgaris, D. Fructosovorans, and A. vinosum.…”