A key question for the understanding of photosynthetic water oxidation is whether the four oxidizing equivalents necessary to oxidize water to dioxygen are accumulated on the four Mn ions of the oxygen-evolving complex (OEC), or whether some ligand-centered oxidations take place before the formation and release of dioxygen during the S 3 → [S 4 ] → S 0 transition. Progress in instrumentation and flash sample preparation allowed us to apply Mn Kβ X-ray emission spectroscopy (Kβ XES) to this problem for the first time. The Kβ XES results, in combination with Mn X-ray absorption near-edge structure (XANES) and electron paramagnetic resonance (EPR) data obtained from the same set of samples, show that the S 2 → S 3 transition, in contrast to the S 0 → S 1 and S 1 → S 2 transitions, does not involve a Mn-centered oxidation. On the basis of new structural data from the S 3 -state, manganese μ-oxo bridge radical formation is proposed for the S 2 → S 3 transition, and three possible mechanisms for the O-O bond formation are presented.

[FeFe]-hydrogenases catalyze the reversible reduction of protons to molecular hydrogen with extremely high efficiency. The active site (“H-cluster”) consists of a [4Fe–4S]H cluster linked through a bridging cysteine to a [2Fe]H subsite coordinated by CN− and CO ligands featuring a dithiol-amine moiety that serves as proton shuttle between the protein proton channel and the catalytic distal iron site (Fed). Although there is broad consensus that an iron-bound terminal hydride species must occur in the catalytic mechanism, such a species has never been directly observed experimentally. Here, we present FTIR and nuclear resonance vibrational spectroscopy (NRVS) experiments in conjunction with density functional theory (DFT) calculations on an [FeFe]-hydrogenase variant lacking the amine proton shuttle which is stabilizing a putative hydride state. The NRVS spectra unequivocally show the bending modes of the terminal Fe–H species fully consistent with widely accepted models of the catalytic cycle.

The organometallic H cluster at the active site of [FeFe]-hydrogenase consists of a 2Fe subcluster coordinated by cyanide, carbon monoxide, and a nonprotein dithiolate bridged to a [4Fe-4S] cluster via a cysteinate ligand. Biosynthesis of this cluster requires three accessory proteins, two of which (HydE and HydG) are radical S-adenosylmethionine enzymes. The third, HydF, is a GTPase. We present here spectroscopic and kinetic studies of HydF that afford fundamental new insights into the mechanism of H-cluster assembly. The thorough kinetic characterization of the GTPase activity of HydF shows that activity can be gated by monovalent cations and further suggests that GTPase activity is associated with synthesis of the 2Fe subcluster precursor on HydF, rather than with transfer of the assembled precursor to hydrogenase. Interestingly, we show that whereas the GTPase activity is independent of the presence of the FeS clusters on HydF, GTP perturbs the EPR spectra of the clusters, suggesting communication between the GTP-and cluster-binding sites. Together, the results indicate that the 2Fe subcluster of the H cluster is synthesized on HydF from a [2Fe-2S] cluster framework in a process requiring HydE, HydG, and GTP.T he reversible reduction of protons, a reaction central to bioenergy and fuel cell applications, is a conceptually simple but chemically challenging reaction. In biology, these reactions occur at unique organometallic metal centers that contain biochemically unusual nonprotein ligands such as carbon monoxide and cyanide. In the case of the [FeFe]-hydrogenase, the site of catalysis is a metal cluster, termed the H cluster, consisting of a [4Fe-4S] cubane bridged by a cysteine thiolate to a 2Fe unit coordinated by carbon monoxide, cyanide, and a bridging dithiolate ligand (Fig. 1) (1-6). The [FeFe]-hydrogenase is of particular interest for bioenergy applications because of its high catalytic rates of proton reduction; however, a limiting factor in its practical utilization is the lack of understanding of the biosynthesis of the organometallic active site cluster. Assembly of a catalytically competent H cluster requires the actions of three hydrogenase-specific accessory proteins, two of which (HydE and HydG) are radical S-adenosylmethionine (SAM) enzymes and the third of which (HydF) is a GTPase (7, 8). These accessory proteins are directed at synthesis of the 2Fe subcluster of the H cluster, which is subsequently transferred to the hydrogenase structural protein (HydA) containing a preformed [4Fe-4S] cluster (9, 10) to produce the active hydrogenase. The detailed stepwise mechanism of H-cluster assembly, as well as the specific roles of and interactions between the three accessory proteins in this assembly process, remains largely unknown. Herein we provide evidence that the 2Fe subcluster of the H cluster is synthesized on HydF from a [2Fe-2S] precursor by the activities of HydE and HydG and that GTP hydrolysis likely plays a role in the assembly of this precursor on HydF.Radical SAM enzymes are charact...