Synthesis of the 2Fe subcluster of the [FeFe]-hydrogenase H cluster on the HydF scaffold

Abstract

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...

CO and CN − syntheses by [FeFe]-hydrogenase maturase HydG are catalytically differentiated events

“…Results reported by Dinis et al (19) and Suess et al (27), along with the observations reported here, suggest that the labile nature of the fifth iron binding may be a requirement for the dissociation of the nascent Fe(CO)x(CN)y unit from HydG and its transfer to the scaffold/carrier HydF (8,9). We speculate that in vivo, Cys, Hcy, or a related ligand (collectively called L) could contribute to the binding stability of this unique ion and could Fig.…”

“…Work from several laboratories has shown that the maturation of the [FeFe] center requires at least three protein maturases: HydF that has GTPase activity and appears to be both a [FeFe] center scaffold and carrier (8,9), HydG that synthesizes CO and CN − from tyrosine (10)(11)(12)(13), and HydE that, by elimination, should be involved in the synthesis of the DTMA bridge (14,15). Both HydE and HydG are members of the large radical S-adenosyl-L-methionine (SAM) protein family (16,17).…”

Mechanism of Proton Transfer in [FeFe]-Hydrogenase from Clostridium pasteurianum

“…[FeFe]-Hydrogenases demonstrate the highest in vitro catalytic turnover among hydrogen-producing enzymes, and there is great interest in elucidating the enzymatic mechanism of these hydrogenases (3,4). Current research has focused primarily on examining the catalytic activity (5,6), active site assembly (7)(8)(9)(10), and irreversible oxygen inactivation of these enzymes (11)(12)(13), but relatively little data are available concerning the intramolecular transport of substrates between the active site and the enzyme surface (14 -16). Proton transfer is an essential component of reversible hydrogen production by [FeFe]-hydrogenases, and biochemical investigation is required for a complete understanding of the enzymatic mechanism.…”

Metallocofactor assembly for [FeFe]-hydrogenases

“…Given the important role for HydF in the complex process of [2Fe] H subcluster assembly, it is perhaps unsurprising that the reported nature of the cluster(s) bound to HydF vary depending on the protein source, chemical reconstitution, heterologous or homologous expression, and co-expression with or without the other maturases (termed HydF EG and HydF ΔEG respectively). There is good agreement that HydF binds a [4Fe-4S] cluster, but variability in the observed state of the second Fe-S cluster: for example, reconstituted TmHydF ΔEG and TnHydF ΔEG likely bear no second cluster [37,38], EPR spectra of unreconstituted CaHydF ΔEG shows a signal resembling a [2Fe-2S] cluster [39,40] and IR spectroscopy suggests multiple diatomic ligands bound to CaHydF EG [40,41]. Models for cluster assembly by HydF agree that the [4Fe-4S] is assembled by housekeeping iron sulfur cluster biosynthetic enzymes.…”

“…Although many unresolved questions remain, using a biosynthetic cocktail (including maturases, substrates and cofactors) to achieve efficient transfer of labelled 57 Fe from HydG to the [2Fe] H subcluster of HydA [16] supports model (ii). Direct interactions between the maturase proteins HydE and HydG with HydF have been demonstrated [29,39] and GTP has been shown to accelerate the dissociation of HydE:HydF and HydG:HydF complexes [40], although the significance of these interactions and the GTPase activity awaits more detailed functional analysis.…”