Compositional and Structural Insights into the Nature of the H-Cluster Precursor on HydF

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 Fe^I-Fe^I 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.