FeFe-Hydrogenase Maturation: Insights into the role HydE plays in dithiomethylamine biosynthesis

摘要

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.