Theses and Dissertations at Montana State University (MSU)
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Item Discovery of key intermediates for radical initiation in PFL-AE(Montana State University - Bozeman, College of Letters & Science, 2020) McDaniel, Elizabeth Claire; Chairperson, Graduate Committee: Joan B. Broderick; This is a manuscript style paper that includes co-authored chapters.Members of the radical S-adenosyl-L-methionine (SAM) enzyme superfamily utilize a [4Fe-4S] cluster and the small molecule, SAM, to generate methionine and the 5'deoxyadenosyl radical (5'-dAdo*). Once formed, the 5'-dAdo* abstracts a hydrogen from substrate allowing for the catalyzation of a wide array of chemistry such as DNA repair, hydrogenase maturation, and anaerobic glucose metabolism. Originally, the 5'-dAdo* was thought to form directly through homolytic cleavage of the S-C5' bond on SAM. In 2016, this mechanism was called into question when a catalytically relevant organometallic intermediate (omega) was discovered in pyruvate formate-lyase activating enzyme (PFL-AE). This intermediate consisted of a 5'-dAdo moiety bound to the unique iron on the PFL-AE [4Fe-4S] cluster through an Fe-C5' bond. The work shown in this thesis provides novel insights into the RS enzyme mechanism considering the newly discovered omega species. Using rapid freeze quench (RFQ) in conjunction with electron paramagnetic resonance (EPR) spectroscopy, omega formation was observed in seven RS enzymes representing the totality of superfamily reaction types. Inspired by the idea that the Fe-C5' bond in omega could undergo photoinitiated homolysis, a unique procedure was developed to generate and capture the long elusive 5'-dAdo* through cryogenic photolysis of reduced PFL-AE and SAM. Isotopic labeling of SAM along with EPR spectroscopy confirmed definitely that this was the long sought after 5'-dAdo*. To better understand RS enzyme bond specificity and the order of intermediate formation, an analogue of SAM, S-3'4'-anhydroadenosyl-L-methionine (anSAM), was employed in RFQ and cryogenic photolysis experiments. By using anSAM, it was shown that the bond cleavage specificity of PFL-AE can changed under appropriate conditions and provided evidence that omega forms first in the radical initiation pathway of RS enzymes. These results have greatly increased our understanding of the RS enzyme mechanism and will help future work designed to utilize the incredible enzymatic potential of this diverse superfamily.Item New insights into radical initiation by radical S-adenosylmethionine enzymes and activation of [FeFe]-hydrogenase(Montana State University - Bozeman, College of Letters & Science, 2020) Impano, Stella; Chairperson, Graduate Committee: Joan B. Broderick; Hao 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.Radical 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.Item Radical S-adenosyl-L-methionine enzymes: radical control and assembly of complex metallocofactors(Montana State University - Bozeman, College of Letters & Science, 2018) Byer, Amanda Shaw; Chairperson, Graduate Committee: Joan B. Broderick; Elizabeth C. McDaniel, Stella Impano, William E. Broderick and Joan B. Broderick were co-authors of the article, 'Mechanistic studies of radical SAM enzymes: pyruvate formate-lyase activating enzyme and lysine 2,3-aminomutase' in the journal 'Methods in enzymology' which is contained within this dissertation.; Masaki Horitani was an author and Krista A. Shisler, Tilak Chandra, Joan B. Broderick and Brian M. Hoffman were co-authors of the article, 'Why nature uses radical S-adenosyl-L-methionine enzymes so widely: electron nuclear double resonance studies of lysine 2,3-aminomutase show the 5'-dADO 'free radical' is never free' in the journal 'Journal of the American Chemical Society' which is contained within this dissertation.; Hao Yang, Elizabeth C. McDaniel, Venkatesian Kathiresan, Stella Impano, Adrien Pagnier, Hope Watts, Carly Denler, Anna Vagstad, Jorn Piel, Kaitlin S. Duschene, Eric M. Shepard, Thomas P. Shields, Lincoln G. Scott, Edward A. Lilla, Kenichi Yokoyama, William E. Broderick, Brian M. Hoffman, and Joan B. Broderick were co-authors of the article, 'New paradigm for radical SAM enzyme reactions: organometallic intermediate Omega is central to catalysis' in the journal 'Journal of the American Chemical Society' which is contained within this dissertation.; Eric M. Shepard was an author and Priyanka Aggarwal, Jeremiah N. Betz, Krista A. Shisler, Robert J. Usselman, Gareth R. Eaton, Sandra S. Eaton, Joan B. Broderick were co-authors of the article, 'Hydrogenase maturase HydF: insights into [2Fe-2S] and [4Fe-4S] cluster communication and hydrogenase activation' in the journal 'Biochemistry' which is contained within this dissertation.; Eric M. Shepard, William E. Broderick and Joan B. Broderick were co-authors of the article, 'Activation of [FeFe]-hydrogenase in the absence of HydG' which is contained within this dissertation.; Donald S. Wright, Michael W. Ratzloff, Yisong Guo, Paul W. King and Joan B. Broderick were co-authors of the article, '[FeFe]-hydrogenase metallocluster assmebly on HydF as influenced by HydG' which is contained within this dissertation.; Amanda Shaw Byer is not the main author of an article which is contained within this dissertation.Electrons, whether from carbon-based radicals or metals, can generate oxidative stress and disease in biological systems; however, when directed properly by a protein, these electrons are responsible for crucial life-sustaining reactions, including photosynthesis, oxygen transport in blood, and nitrogen fixation. Beneficial use of radicals and metallocofactors is abundant in nature, and both are essential in one of the largest superfamilies in biology - the radical SAM (RS) enzyme superfamily. Found in all kingdoms of life, RS enzymes contribute to critical processes such as DNA repair, complex metallocluster assembly, and vitamin synthesis. Understanding how metalloenzymes, such as RS enzymes, control electron flow is critical for comprehending biological system functionality and potentially improving productivity through rational design. This work examines radical control in RS enzyme mechanism and then expands scope to consider RS enzyme contribution to assembly of the complex metallocluster (Hcluster) of [FeFe]-hydrogenase. Focusing in on the fundamental chemistry of RS enzyme radical initiation, this work investigated intermediate states in 5'deoxyadenosyl radical formation by: 1) slowing the radical reaction with a SAM analogue, anSAM, and 2) swiftly stopping catalysis via rapid freeze quench techniques. Employing primarily EPR and ENDOR spectroscopies, two intermediate states were characterized: 1) an analogue of the 5'-deoxyadenosyl radical, formed from anSAM, and 2) an organometallic intermediate, Omega, formed during reaction with SAM. To probe how certain RS enzymes (HydE and HydG) contribute to build the 2Fe H-cluster subcluster precursor on the [FeFe]-hydrogenase scaffold HydF, FeS cluster intermediate states were analyzed using UV-Vis, EPR, FTIR, CD, Mossbauer spectroscopies and gas chromatography. These results demonstrate: 1) HydF initially binds a [4Fe-4S] and a [2Fe-2S] cluster, 2) HydG contributes small molecule diatomics and perturbs the [2Fe-2S] cluster environment, 3) HydE can generate a subcluster precursor on HydF capable of generating catalytically active HydA, and 4) the HydF dimer, not tetramer, delivers the 2Fe H-cluster subcluster precursor for activation. Collectively, this thesis illuminates key mechanistic states RS enzymes use to productively control the 5'deoxyadenosyl radical during catalysis and identifies [FeFe]-hydrogenase H-cluster precursor intermediates suggesting RS enzyme sequentiality.Item Spectroscopic investigations into the active site structure and the mechanisms of radical SAM enzymes(Montana State University - Bozeman, College of Letters & Science, 2016) Shisler, Krista Ann; Chairperson, Graduate Committee: Joan B. Broaderick; Joan B. Broderick was a co-author of the article, 'Emerging themes in radical SAM chemistry' in the journal 'Current opinion in structural biology' which is contained within this dissertation.; Joan B. Broderick was a co-author of the article, 'Glycyl radical activating enzymes: structure, mechanisms and substrate interactions' in the journal 'Archives of biochemistry and biophysics' which is contained within this dissertation.; Masaki Horitani, Brian M. Hoffman and Joan B. Broderick were co-authors of the article, 'EPR and ENDOR analysis of small molecules inducing valence localization in PFL-AE' submitted to the journal 'Journal of the American Chemical Society' which is contained within this dissertation.; Rachel U. Hutcheson, Kaitlin S. Duschene, Adam V. Crain, Ashley Rasmussen, Jian Yang, Jessica L. Vey and Joan B. Broderick were co-authors of the article, 'The activation of the radical SAM enzyme pyruvate formate lyase activating enzyme is stimulated by potassium' submitted to the journal 'Biochemistry' which is contained within this dissertation.; Masaki Horitani, Kaitlin Duschene, Rachel U. Hutcheson, Amy Marts, George Cutsail III, William Broderick, Brian M. Hoffman and Joan B. Broderick were co-authors of the article, 'A rapid freeze quench ENDOR study of an organometallic radical intermediate in PFL-AE' submitted to the journal 'Journal of the American Chemical Society' which is contained within this dissertation.; This dissertation contain one article of which Krista Ann Shisler is not the main author.The radical S-adenosyl-L-methionine (SAM) superfamily of enzymes carry out diverse and complex reactions through generation of a 5'-deoxyadenosyl (5'-dAdo·) radical followed by transfer to substrate. These enzymes contain a [4Fe-4S] cluster which binds and transfers an electron to SAM. The exact mechanism of 5-dAdo· generation is unknown and the studies herein provide further investigation into pyruvate formate lyase activating enzyme (PFL-AE) and lysine 2,3-aminomutase (LAM) pre and post SAM cleavage. To understand the active site of PFL-AE prior to SAM cleavage, cation and small molecule effects were examined by electron paramagnetic resonance (EPR) and electron nuclear double resonance (ENDOR) spectroscopies. Previously, PFL-AE had been observed to contain a valence localized cluster in the presence of small molecules and this work used EPR and ENDOR spectroscopy to further probe the effects of these molecules. These studies determined that these molecules do not directly bind the cluster but rather an H xO species occupies the unique Fe site. The crystal structure of PFL-AE revealed a cation site and to probe this site, EPR and ENDOR spectroscopies were employed. Monovalent cations stimulated PFL-AE activity, with the greatest activity in the presence of potassium. The identity of the cation perturbed the EPR signal of PFL-AE which was more pronounced in the presence of SAM. ENDOR spectroscopy determined that SAM coordination differed depending on the monovalent cation. Due to its high reactivity, 5'-dAdo· has never been spectroscopically observed. In order to examine any intermediate states, a SAM analog and rapid freeze quench (RFQ) techniques were employed in conjunction with EPR and ENDOR spectroscopies. LAM can cleave the SAM analog, S-3',4'-anhydroadenosyl-L-methionine, to produce a stable allylic radical which was coupled with isotopically labeled lysine for ENDOR analysis. It was determined that radical generation is highly controlled with little movement towards its substrate upon 5'-dAdo· production. During RFQ techniques on PFL-AE, an organometallic intermediate species was observed. To probe this intermediate, isotopically labeled SAM and an 57Fe labeled cluster were coupled with the unknown paramagnetic species. It was determined that this intermediate was an unprecedented organometallic Fe-adenosyl bound species post SAM cleavage.Item Mechanistic and spectroscopic investigations of the radical SAM maturases HydE and HydG for [FeFe]-hydrogenase(Montana State University - Bozeman, College of Letters & Science, 2015) Betz, Jeremiah Nathanael; Chairperson, Graduate Committee: Joan B. Broderick; Nicholas W. Boswell, Corey J. Fugate, Gemma L. Holliday, Eyal Akiva, Anna G. Scott, Patricia C. Babbitt, John W. Peters, Eric M. Shepard and Joan B. Broderick were co-authors of the article, '[FeFe]-hydrogenase maturation: insights into the role HydE plays in dithiomethylamine biosynthesis' in the journal 'Biochemistry' which is contained within this thesis.; Thesis contains article(s) of which Jeremiah Nathanael Betz is not the main author.While biochemical, spectroscopic, and analytical investigations helped classify multiple phylogenetically distinct hydrogenases it was not until 2004 that Peters et al. gave the world a look at the non-proteinaceous component of the active site of a hydrogenase enzyme. The active site (H-cluster) of [FeFe]-hydrogenase was found to possess a typical [4Fe-4S] cluster bridged by the sulfur of a cysteinyl group to an iron of a uniquely decorated 2Fe subcluster that serves as the site of molecular hydrogen synthesis and oxidation. The subcluster contains two irons bridged by a dithiomethylamine (DTMA) group and a carbon monoxide ligand. In addition each iron is coordinated by a carbon monoxide and cyanide ligand. Posewitz et al. in 2004 were the first to shed light on the syntheses of these non-proteinaceous ligands when through an insertional mutagenesis study of a hydrogen producing green alga they found two radical SAM enzymes, HydE and HydG, that were required for the maturation of [FeFe]- hydrogenase. HydG has been extensively studied and been shown to produce the diatomic ligands of the H-cluster from tyrosine. In this work the substrate specificity and active site of HydG was investigated. These investigations led to a refinement of the location and mechanism of H-atom abstraction of the substrate HydG and support the identity of the C-terminal FeS cluster as a [4Fe-4S] cluster that is responsible in the later steps of diatomic production. While several crystal structures of HydE have been published, the work reported herein is the first to propose a substrate and reaction mechanism for HydE. The results point to commonly biologically available low molecular weight thiols such as L-cysteine, L-homocysteine, and mercaptopyruvate as likely substrates. More recent work has implicated mercaptopyruvate as the substrate given glyoxylate was produced under turnover conditions. Our proposed mechanism involves formation of thioformaldehyde from mercaptopyruvate. Two thioformaldehyde units may be condensed with ammonia forming the DTMA precursor. While many details remain unsolved regarding the maturation of [FeFe]-hydrogenase, our findings regarding HydE and HydG are important steps forward in the understanding of biological catalysts of hydrogen production.Item Mechanism of diatomic ligand biosynthesis by radical s-adenosylmethionine [FeFe]-hydrogenase maturase HydG(Montana State University - Bozeman, College of Letters & Science, 2014) Duffus, Benjamin Richard; Shourjo Ghose, John W. Peters and Joan B. Broderick were co-authors of the article, 'Reversible H atom abstraction at the tyrosine phenol position catalyzed by the radical SAM enzyme HydG' submitted to the journal 'Journal of the American Chemical Society' which is contained within this thesis.; Simon J. George, Aubrey D. Scott, Eric M. Shepard, Kaitlin D. Duschene, Stephen P. Cramer, John W. Peters and Joan B. Broderick were co-authors of the article, 'Defining a basis for diatomic ligand product binding to the radical SAM enzyme HydG' submitted to the journal 'Biochemistry' which is contained within this thesis.; Rebecca C. Driesener, Eric M. Shepard, Peter L. Roach, John W. Peters and Joan B. Broderick were co-authors of the article, 'HydG carbon monoxide formation stoichiometry: the role of phosphate in diatomic ligand biosynthesis' submitted to the journal 'Biochemistry' which is contained within this thesis.; Eric M. Shepard, John W. Peters and Joan B. Broderick were co-authors of the article, 'Effector and intermediate molecule interaction with radical SAM [FeFe]-hydrogenase maturase HydG' submitted to the journal 'Biochemistry' which is contained within this thesis.; Eric M. Shepard, John W. Peters and Joan B. Broderick were co-authors of the article, 'Delineating H atom abstraction in HydG catalysis with tyrosine analogues and site-directed mutagenesis' submitted to the journal 'Biochemistry' which is contained within this thesis.Iron-sulfur (Fe-S) clusters are ubiquitous in biology, and serve as catalysts in a vast array of chemical transformations that comprise central metabolic reactions and small molecule interconversions. Complex Fe-S clusters such as the [FeFe]-hydrogenase "Hcluster" cofactor are part of a distinct subgroup of metalloenzymes that have evolved from reduced Fe-S mineral phases, as the H-cluster catalyzes H-H bond formation through reduction of protons with electrons. Biosynthesis of this cofactor is unique in its involvement of two radical S-adenosylmethionine (SAM) enzymes HydG and HydE, and a scaffold GTPase HydF. Together, these proteins synthesize a unique Fe-S cluster that coordinates a bridging dithiolate ligand as well as two CN- and three CO ligands However, many mechanistic details relating to the biosynthesis are not well known. In this work, the radical SAM enzyme HydG has been shown to synthesize CO, CN-, and pcresol through a radical-initiated fragmentation of the substrate tyrosine. The catalytic mechanism is complex, because an accessory C-terminal Fe-S cluster is required for catalysis. The exact role of this cluster in the biosynthetic mechanism is unresolved, but is proposed to serve a modular role as a potential scaffold for diatomic ligand synthesis. To understand the catalytic mechanism, a combined biochemical and spectroscopic approach was applied. In this work, it is shown that the C-terminal Fe-S cluster is essential for the formation of both CO and CN- products. Spectral characterization of the enzyme has shown the formation of diatomic ligand products that are bound to the coordinated Fe-S clusters. Also, an H atom abstraction profile of HydG has been recently characterized to provide insight to the involvement of the 5'-deoxyadenosyl radical in catalysis. Further mechanistic insight into catalysis has also been investigated through site-directed mutagenesis and through using substrate analogues. The work presented as a whole, by establishing parallels to the radical SAM enzyme superfamily in character to biosynthesis, reveals unifying themes in complex metal cluster assembly related to radical-initiated modification of ordinary Fe-S clusters via product organometallic complex formation.Item Investigating the structural and mechanistic paramters of two radical SAM enzymes, spore photoproduct lyase and HydE(Montana State University - Bozeman, College of Letters & Science, 2013) Ghose, Shourjo; Chairperson, Graduate Committee: Joan B. Broderick; Jonathan K. Hilmer, Kaitlin Duschene, Brian Bothner and Joan B. Broderick were co-authors of the article, 'Solution phase dyanamics of the DNA reoair enzyme spore photoproduct lyase as probed by H/D exchange' submitted to the journal 'FEBS letters' which is contained within this thesis.; Nicholas Boswell, Eric M. Shepard, John W. Peters and Joan B. Broderick were co-authors of the article, 'Identification and quantitation of a putative substrate for [FE FE]-hydrogenase maturase enzyme HydE' submitted to the journal 'Biochemistry' which is contained within this thesis.; Eric M. Shepard, John W. Peters and Joan B. Broderick were co-authors of the article, 'Mechanistic insights into HydE catalysis' submitted to the journal 'Biochemistry' which is contained within this thesis.The resistance of Bacterial spores to UV radiation makes them a causative agent in many diseases and poses a threat to humans and animals alike. This unique resistance stems from the repair of a thymine dimer, 5-thyminyl-5,6-dihydrothymine (spore photoproduct, or SP)on exposure to UV irradiation. During the early stages of germination, this SP is repaired by an enzyme, spore photoproduct lyase (SPL) into two thymines. SPL is a member of the radical SAM superfamily of enzymes and requires S-adenosylmethionine (SAM) and a [4Fe-4S]1+/2+ cluster to perform its catalysis. The first part of this dissertation is dedicated towards understanding the solution phase dynamics of this protein on binding with its substrate and co-factor via hydrogen deuterium exchange. Analyses of the effects of SAM binding to SPL indicate that the protein does go through a conformational change localized around its active site. We have also demonstrated that the concomitant binding of SAM and dinucleotide SP contributes more significantly to the active site stabilization than what is observed with just SAM binding. Moreover we have provided initial evidence that the SPL might be utilizing the deformation of the phosphodiester back bone of SP to recognize, bind and initiate catalysis. We have unequivocally demonstrated that the catalytic [4Fe-4S] cluster plays a significant role in substrate/cofactor binding most likely due to the stabilization of the 8 residue loop region it resides on. The second part of this dissertation is focused towards understanding the role of maturase proteins in the assembly of the active site of [FeFe]- hydrogenase. The assembly and biosynthesis of the H-cluster requires three accessory enzymes HydE, HydG and HydF. Herein show that HydE utilizes cysteine as a substrate. We have also shown through LCMS and specifically deuterium labeled substrate, that catalysis is initiated via a H atom abstraction from the beta carbon of cysteine. Our investigations into the mechanism of HydG mediated turnover of tyrosine reveal that catalysis is initiated via a single H atom abstraction from the phenolic position of the substrate. Taken together we believe that our investigations have provided some critical insights into specific roles of these enzymes.Item Interactions and electron transfer involved in pyruvate formate-lyase activation(Montana State University - Bozeman, College of Letters & Science, 2013) Crain, Adam Vernon; Chairperson, Graduate Committee: Joan B. Broderick; Kaitlin S. Duschene, John W. Peters and Joan B. Broderick were co-authors of the article, 'Iron sulfur clusters, S-adenosylmetionine enzymes, and their role in hydrogenase maturation' which is contained within this thesis.; Joan B. Broderick was a co-author of the article, 'Flavodoxin cofactor binding induces structural changes that are required for protein-protein interactions with NADP+ oxidoreductase and pyruvate formate-lyase activating enzyme' which is contained within this thesis.; Joan B. Broderick was a co-author of the article, 'Pyruvate formate-lyase: protein-protein interactions and activation by pyruvate formate-lyase activating enzyme' which is contained within this thesis.; Stephanie J. Maiocco, Sean J. Elliot and Joan B. Broderick were co-authors of the article, 'Elucidating the role of cation binding in PFL-AE' which is contained within this thesis.; Martina D. Van Hoy and Joan B. Broderick were co-authors of the article, 'Pyruvate:flavodoxin oxidoreductase is the electron donor for pyruvate formate-lyase activating enzyme' which is contained within this thesis.Pyruvate formate-lyase activating enzyme (PFL-AE) is one of the best-characterized members of the radical S-adenosyl-L-methionine (SAM) superfamily. The radical SAM enzymes utilize an iron-sulfur cluster and SAM to catalyze a diverse set of reactions such as vitamin synthesis, enzyme activation, DNA repair, and sulfur insertion to name a few. PFL-AE contains one [4Fe-4S] cluster coordinated by cysteine residues from a canonical CX3CX2C radical SAM motif. Iron-sulfur cluster-initiated reductive cleavage of S-adenosylmethionine results in a highly reactive 5'-deoxyadenosyl radical that abstracts a pro-S hydrogen from glycine 734 on PFL creating a stable glycyl radical. PFL utilizes this glycyl radical to catalyze the reaction pyruvate + CoA [] formate + acetyl-CoA, thereby providing the main source of acetyl-CoA for the citric acid cycle under anaerobic conditions. We have undertaken experiments using circular dichroism and isothermal titration calorimetry to characterize interactions between flavodoxin (Fld) and its cofactor (FMN). These experiments show that cofactor binding significantly increases flavodoxin stability and structure, which is required for protein-protein interactions. Anaerobic surface plasmon resonance experiments were used to provide insight into protein-protein interactions between the enzymes involved in PFL activation and in all cases, the proteins interact with low micromolar affinity. SAM binding experiments with PFL-AE were performed in the presence and absence of PFL, which demonstrate that PFL binding to PFL-AE does not alter SAM binding affinity for PFL-AE. PFL activation studies using PFL-AE in the presence of PFL substrates/analogues show that they are not required for PFL activation, however they do play a large role in activation and their inclusion resulted in 3.7 fold higher glycyl radical concentrations. In vivo concentrations were calculated for proteins and small molecules involved in PFL activation and activity in E. coli to provide a context for our measured equilibrium constants and to determine the amount of bound protein in vivo. Activity assays and UV-vis electron transfer assays show that pyruvate:flavodoxin oxidoreductase (PFOR) is capable of activating the PFL system. The aggregate data suggests that electron transfer from Fld to PFL-AE only occurs when SAM and PFL are bound to PFL-AE.Item Mechanistic and spectroscopic investigations of pyruvate formate-lyase activating enzyme(Montana State University - Bozeman, College of Letters & Science, 2012) Hutcheson, Rachel Ann Udelhoven; Chairperson, Graduate Committee: Joan B. Broderick; Kaitlin S. Duschene, Adam V. Crain, Ashley Rasmussen, Jian Yang, Jessica L. Vey, Catherine L. Drennan and Joan B. Broderick were co-authors of the article, 'The activation of pyruvate formate-lyase activating enzyme is stimulated by K+' which is contained within this thesis.; Kaitlin S. Duschene, Adam V. Crain, Yi Peng, J. Timothy Sage and Joan B. Broderick were co-authors of the article, 'NRVS reveals changes in the PFL-AE cluster upon SAM and substrate analog binding' which is contained within this thesis.; Joan B. Broderick was a co-author of the article, 'Radical SAM enzymes in methylation and mehtylthiolation' in the journal 'Metallomics' which is contained within this thesis.Radical S-adenosylmethionine (SAM) enzymes are a large and rapidly growing superfamily composed of thousands of members catalyzing a wide diversity of reactions by utilizing a reduced [4Fe-4S] ¹ + cluster and SAM to create a 5'-deoxyadenosyl radical capable of initiating controlled radical chemistry in important and difficult biochemical reactions. The prevalence of radical SAM enzymes in all kingdoms of life underscores the central role played by these enzymes. For the vast majority of putative radical SAM enzymes little is known regarding the reaction catalyzed or the mechanism of catalysis. Nevertheless, it is possible to gain insight into these enzymes from the radical SAM enzyme pyruvate formate-lyase activating enzyme (PFL-AE), which catalyzes the formation of a catalytically essential glycyl (G734) radical of pyruvate formate-lyase (PFL). The studies presented herein provide further understanding and characterization of PFL-AE as well as other radical SAM enzymes. The relevance and effect of the monovalent cation found in the active site of PFL-AE upon further analysis of the crystal structure was probed using coupled enzyme activity assays. Five different monovalent cations, Na +, K +, NH 4 +, Rb +, and Cs +, were investigated by calculating the specific activity of PFL-AE in the presence of each. PFL-AE was active in the presence of all tested cations, with specific activities correlating with cation size. Nuclear resonance vibrational spectroscopy performed on PFL-AE with an ⁵⁷ Fe labeled cluster showed a enzyme stiffening around the cluster and elongation of Fe-S bonds upon substrate and substrate analog binding. Rapid freeze-quench was used to mix PFL-AE with PFL and SAM on a millisecond time scale. The resulting samples were analyzed by electron paramagnetic resonance, which revealed a newly observed radical intermediate. To attempt characterization of this radical intermediate, electron nuclear double resonance spectroscopy (ENDOR) was used with site-specifically labeled SAM. The ENDOR signal detected was too weak to be analyzed; however, other labeled SAM molecules will be used in the future. To help further expand knowledge of radical SAM enzymes, an initial characterization of a putative methylthiotransferase (a subclass of the radical SAM superfamily) was undertaken. Results indicated that the enzyme methylthiolated a ribosomal small protein and not tRNA.