Chairperson, Graduate Committee: Joan B. BroderickImpano, StellaHao Yang, Adrien Pagnier, Richard Jodts, Ryan Swimley, Eric M. Shepard, Sarah M. Hill, Christopher D. James, William E. Broderick, Brian M. Hoffman and Joan B. Broderick were co-authors of the article, 'Photolytic cleavage of S-adenosylmethionine' which is contained within this dissertation.Eric M. Shepard, Hao Yang, Adrien Pagnier, Ryan Swimley, Emma Dolen, William E. Broderick, Brian M. Hoffman and Joan B. Broderick were co-authors of the article, 'Generation of an ethyl radical trapped in active sites of [FeFe]-hydrogenase maturase enzymes HydE AND HydG' which is contained within this dissertation.Eric M. Shepard, Hao Yang, Jeremiah N. Betz, Adrien Pagnier, William E. Broderick, Brian M. Hoffman and Joan B. Broderick were co-authors of the article, 'EPR and ENDOR spectroscopic evidence of an ammonium binding site in HydE' which is contained within this dissertation.Adrien Pagnier, Eric M. Shepard, William E. Broderick and Joan B. Broderick were co-authors of the article, 'Investigation into all the necessary components required for [FeFe]-hydrogenase H-cluster maturation' which is contained within this dissertation.Dissertations contains two articles of which Stella Impano is not the main author.2022-05-132022-05-132020https://scholarworks.montana.edu/handle/1/16734Radical S-adenosylmethionine (SAM) enzymes harbor a [4Fe-4S] cluster in their active sites that coordinates a catalytically relevant small molecule SAM. During catalysis the S-5'C bond of SAM is reductively cleaved to generate a 5'-deoxyadenosyl radical that subsequently abstracts an H atom from substrate, allowing functionally diverse reactions to be achieved. Trapping of the 5'-deoxyadenosyl radical intermediate during turnover had proven difficult likely due to the formation of omega intermediate resulting from the oxidative addition of the 5'-deoxyadenosyl radical to the unique iron of the cluster. Recently, our laboratory showed that this elusive 5'-deoxyadenosyl can be liberated, captured, and characterized, in the absence of substrate, via photoinduced electron transfer (ET)-mediated reductive cleavage of SAM. Further, photolysis of [4Fe-4S] +-SAM complexes in different radical SAM enzymes revealed that the regioselective bond cleavage of SAM is dependent on the active site environment where either a 5'-deoxyadenosyl or a *CH 3, depending on the enzyme. When Sadenosyl- ethionine is used in place of SAM in the [4Fe-4S] +-SAM complex of HydE or HydG an ethyl radical is trapped. In either case, annealing of the methyl and ethyl radicals yields corresponding omega-like species, omega M and omega E, respectively. Functionally, HydE and HydG work together with a third protein HydF, to synthesize the H-cluster of [FeFe]-hydrogenase enzymes. HydG lyses tyrosine to generate CO and CN - ligands of the diiron core of the H-cluster, while the role and substrate of HydE are yet to be elucidated; however, it is hypothesized that this enzyme is responsible for dithiomethylamine (DTMA) bridge assembly. Our hypothesis is that HydE uses ammonium as a co-substrate and we propose that this polyatomic ion condenses with two CH 2S- like species to assemble the DTMA. We demonstrate for the first time via EPR and ENDOR spectroscopic techniques that HydE harbors an ammonium binding site; this NH 4 + would be stored in the active site of HydE prior to DTMA synthesis. Additionally, through in vitro [FeFe]-hydrogenase assays, we investigate what component of the essential E. coli lysate is required for H-cluster assembly. Results from this work suggest that the Hyd maturases are not the only proteins needed for H-cluster biosynthesis.enAdenosylmethionineEnzymesHydrogenaseCharge exchangePhotochemistryDissertationCopyright 2020 by Stella Impano