HydE and HydG are radical S-adenosylmethionine enzymes required for the maturation of [FeFe]-hydrogenase (HydA) and produce the non-protein organic ligands characteristic of its unique catalytic cluster. The catalytic cluster of HydA (the H-cluster) is a typical [4Fe-4S] cubane bridged to a 2Fe-subcluster that contains two carbon monoxide, three cyanide, and a bridging dithiomethylamine as ligands. While recent studies have shed light on the nature of diatomic ligand biosynthesis by HydG, little information exists on the function of HydE. Herein, we present biochemical, spectroscopic, bioinformatics, and molecular modeling data that together map the active site and provide significant insight into the role of HydE in H-cluster biosynthesis. Electron paramagnetic resonance and UV-visible spectroscopic studies demonstrate that reconstituted HydE binds two [4Fe-4S] clusters and copurifies with S-adenosyl-L-methionine. Incorporation of deuterium from D2O into 5’-deoxyadenosine, the cleavage product of S-adenosyl-L-methionine, coupled with molecular docking experiments suggests that the HydE substrate contains a thiol functional group. This information, along with HydE sequence similarity and genome context networks, have allowed us to redefine the presumed mechanism for HydE away from BioB-like sulfur insertion chemistry; these data collectively suggest that the source of the sulfur atoms in the dithiomethylamine bridge of the H-cluster are likely derived from HydE’s thiol containing substrate.

Nitrogen-fixing organisms perform dinitrogen reduction to ammonia at an Fe-M (M = Mo, Fe, or V) cofactor (FeMco) of nitrogenase. FeMco displays eight metal centers bridged by sulfides and a carbide having the MFe7S8C cluster composition. The role of the carbide ligand, a unique motif in protein active sites, remains poorly understood. Toward addressing how the carbon bridge affects the physical and chemical properties of the cluster, we isolated synthetic models of subsite MFe3S3C displaying sulfides and a chelating carbyne ligand. We developed synthetic protocols for structurally related clusters, [Tp*M’Fe3S3X]n−, where M’ = Mo or W, the bridging ligand X = CR, N, NR, S, and Tp* = Tris(3,5-dimethyl-1-pyrazolyl)hydroborate, to study the effects of the identity of the heterometal and the bridging X group on structure and electrochemistry. While the nature of M’ results in minor changes, the chelating, μ3-bridging carbyne has a large impact on reduction potentials, being up to 1 V more reducing compared to nonchelating N and S analogs.

Assembly of an active [FeFe]-hydrogenase requires dedicated maturation enzymes that generate the active-site H-cluster: the radical SAM enzymes HydE and HydG synthesize the unusual non-protein ligands - carbon monoxide, cyanide, and dithiomethylamine - while the GTPase HydF serves as a scaffold for assembly of the 2Fe subcluster containing these ligands. In the current study, enzymatically cluster-loaded HydF ([2Fe]F) is produced by co-expression with HydE and HydG in an Escherichia coli host followed by isolation and examination by FTIR and EPR spectroscopy. FTIR reveals the presence of well-defined terminal CO and CN- ligands; however, unlike in the [FeFe]-hydrogenase, no bridging CO is observed. Exposure of this loaded HydF to exogenous CO or H2 produces no significant changes to the FTIR spectrum, indicating that, unlike in the [FeFe]-hydrogenase, the 2Fe cluster in loaded HydF is coordinatively saturated and relatively unreactive. EPR spectroscopy reveals the presence of both [4Fe-4S] and [2Fe-2S] clusters on this loaded HydF, but provides no direct evidence for these being linked to the [2Fe]F. Using the chemical reactivity and FTIR data, a large collection of computational models were evaluated. Their scaled quantum chemical vibrational spectra allowed us to score various [2Fe]F structures in terms of their ability to reproduce the diatomic stretching frequencies observed in the FTIR experimental spectra. Collectively, the results provide new insights that support the presence of a diamagnetic, but spin-polarized FeI-FeI oxidation state for the [2Fe]F precursor cluster that is coordinated by 4 CO and 2 CN- ligands, and bridged to an adjacent iron-sulfur cluster through one of the CN- ligands.