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...