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Item Biochemical characterization of the [FeFe]-hydrogenase maturation protein HydE and identification of the substrate(Montana State University - Bozeman, College of Letters & Science, 2011) Boswell, Nicholas William Bradford; Chairperson, Graduate Committee: Joan B. BroderickHydrogenases catalyze the reversible reduction of protons using complex metal clusters with unusual ligands. The catalytic center of the [FeFe]-hydrogenases is called the H-cluster, and is characterized by a [4Fe-4S] cluster connected via a cysteine thiolate to a 2Fe subcluster coordinated by carbon monoxide and cyanide ligands as well as a bridging dithiolate. Assembly of the H-cluster is carried out by three hydrogenase maturation proteins: HydE, HydF, and HydG. HydF is a GTPase and has been implicated to serve as a scaffold for assembly of the 2Fe subcluster of the H-cluster. HydE and HydG are radical S-adenosylmethionine (SAM) enzymes and thus are thought to utilize reductive cleavage of SAM to initiate radical chemistry. HydG has been shown to catalyze the formation of the carbon monoxide and cyanide ligands of the H-cluster utilizing tyrosine as a substrate. HydE, therefore, has been proposed to be responsible for biosynthesis of the dithiolate ligand of the H-cluster. The aim of this study was to biochemically characterize active, Fe-S reconstituted HydE and to identify the substrate of this radical SAM enzyme. Questions to be studied also included studying the role of HydE in H-cluster maturation. This study used protein purified from recombinant E. coli. The purified protein was chemically reconstituted with iron and sulfide, and used for spectroscopic characterization and HPLC based activity assays. Colorimetric assays were also used for protein characterization and to test for the consumption of substrate. The results indicate that cysteine is likely the substrate of HydE. Activity assays show that HydE- catalyzed SAM cleavage is stimulated in the presence of cysteine, and HydF purified from different genetic backgrounds shows a spectroscopic shift in the lambda max when both HydE and cysteine are present during growth. Spectroscopic characterization confirms that HydE is an Fe-S containing radical SAM enzyme and that cysteine may be a substrate during [FeFe]-hydrogenase H-cluster maturation.