Theses and Dissertations at Montana State University (MSU)
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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 Characterization of CpI and CpII [FeFe]-hydrogenases reveals properties contributing to catalytic bias(Montana State University - Bozeman, College of Letters & Science, 2016) Artz, Jacob Hansen; Chairperson, Graduate Committee: John W. Peters; Dissertation contains two articles of which Jacob Hansen Artz is not the main author.; David W. Mulder, Michael W. Ratzloff, Saroj Poudel, Axl X. LeVan, S. Garrett Williams, Michael W. W. Adams, Anne K. Jones, Eric S. Boyd, Paul W. King and John W. Peters were co-authors of the article, 'Potentiometric EPR spectral deconvolution of CPI [FeFe]-hydrogenase reveals accessory cluster properties' submitted to the journal 'Journal of the American Chemical Society' which is contained within this thesis.; David W. Mulder, Michael W. Ratzloff, Saroj Poudel, Axl X. LeVan, Michael W. W. Adams, Eric S. Boyd, Paul W. King, and John W. Peters were co-authors of the article, 'EPR and FTIR spectroscopy provides insights into the mechanism of [FeFe]-hydrogenase CPII' submitted to the journal 'Journal of the American Chemical Society' which is contained within this thesis.The need for food, fuel, and pharmaceuticals has been increasing at a growing rate as the world's population increases and lifestyles improve. All of these needs are highly energy dependent, and, to a significant degree, rely on an inefficient use of fossil fuels. In order to break free of this dependence, new understanding is required for how to efficiently generate the products humanity needs. Here, a model system of two closely related [FeFe]-hydrogenases, CpI and CpII, is employed in order to understand how biology is able to efficiently control the formation of reduced products, in order to further delineate the limits of control, and the extent to which biology may be co-opted for technological needs. CpI, one of nature's best catalysts for reducing protons to hydrogen gas, is compared to CpII, which functions catalytically to oxidize hydrogen to protons and electrons. Oxygen sensitivity, midpoint potentials, catalytic mechanisms, and catalytic bias are explored in-depth using electron paramagnetic resonance, Fourier Transform Infrared spectroscopy, and protein film voltammetry. CpI and CpII have been found to function under different metabolic conditions, and key amino acids influencing their distinct behavior have been identified. The conduit arrays of hydrogenases, which direct electrons to or from the active site, have been found to have distinct midpoint potentials in CpII compared to CpI, effectively reversing the favored electron flow through CpII in comparison to CpI. In order to probe the contributions of the protein framework on catalysis, analysis of site-specific amino acid substituted variants have been used to identify several determinants that affect the H-cluster environment, which contributes to the observed differences between CpI and CpII. This has resulted in a deeper understanding of the hydrogenase model system and the ability to directly influence catalytic bias. Thus, the work presented here represents key progress towards developing unidirectional catalysts, and demonstrates the possibility of targeted, rational design and implementation of unidirectional catalysts.Item Biological redesign of virus particles for a new era of catalytic materials(Montana State University - Bozeman, College of Letters & Science, 2016) Jordan, Paul Campion; Chairperson, Graduate Committee: Trevor Douglas; Dustin P. Patterson, Kendall N. Saboda, Ethan J. Edwards, Heini M. Miettinen, Gautam Basu, Megan C. Thieleges and Trevor Douglas were co-authors of the article, 'Self-assembling biomolecular catalysts for hydrogen production' in the journal 'Nature chemistry' which is contained within this dissertation.; Joseph C-Y Wang, Ethan J. Edwards, Heini M. Miettinen, Amanda L. Le Sueur, Megan C. Thielges , Adam Zlotnick and Trevor Douglas were co-authors of the article, 'Redesign of a virus particle for NADH-driven hydrogen production' which is contained within this dissertation.; This dissertation contains one article of which Paul Campion Jordan is not the main author.Biology has designed a suite of compartments and barriers that confine fundamental biochemical reactions. Such barriers include the membrane-bound organelles but also a suite of protein-based compartments that architecturally and chemically integrate catalytic processes. These compartments co-polymerize from multiple protein subunits to form polyhedral structures that spatially separate enzymatic processes. Protein compartments confine volatile intermediates, trap toxic reaction products, and co-localize multiple enzymatic processes for catalytic enhancements. The protein-based compartments represent, advantageously, a combination of form and function that has inspired the synthesis of new, designer materials. The self-assembly of cage-like structures, the structures of which are reminiscent of the compartments, has been used for the directed encapsulation of active enzymes. We have used the capsid from bacteriophage P22, as a nanocontainer for directing the encapsulation of a variety of gene products, including active enzymes. The P22 capsid assembles from a coat protein (CP) and a scaffold protein (SP) which templates its assembly. Using the simplicity of the P22 expression system, a strategy was developed and implemented for the directed encapsulation of an active, [NiFe] hydrogenase. We hypothesized and proved the enzyme active site needed to be matured by accessory proteins found within the expression host. A two plasmid expression system was designed, where the hydrogenase cargo was under the control of a different inducer than the P22 CP. The [NiFe]-hydrogenase is a heterodimer and each enzyme subunit was fused to different SP. The resultant packaging of the two SP fusions, with the hydrogenase large and small subunits fused to them stabilized a weak heterodimeric structure. Remarkably, the stabilizing effects of the capsid allowed us to probe the infrared signatures associated with the hydrogenase active site. Finally, the progress made here in developing a virus capsid for H2 production left room to build increased complexity into the P22-Hydrogenase system while also taking inspiration from the innate, biological function of the hydrogenase. We incorporated a cytochrome/cytochrome reductase pair to drive H 2 production using NADH. These designs, built at the molecular level, represent inherently renewable catalysts that pave the way for a new era of catalytic materials synthesized entirely by biology.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 Characterization of the [FeFe]-hydrogenase : toward understanding and implementing biohydrogen production(Montana State University - Bozeman, College of Letters & Science, 2014) Swanson, Kevin Daniel; Chairperson, Graduate Committee: John W. PetersHydrogen may provide an avenue for a clean renewable fuel source, yet the methods to produce hydrogen are either extremely energy intensive, rely on fossil fuels, or require expensive noble metal catalysts. Biology may hold the keys necessary to unlocking new technologies that could change how hydrogen is produced. Microbial processes also produce hydrogen and harbor enzymes that carryout the reversible reduction of protons to hydrogen gas. These enzymes are capable of producing hydrogen at high rates comparable to platinum catalysts, but biological hydrogen catalysts can produce hydrogen using abundant elements carbon, oxygen, nitrogen, sulfur, iron, nickel and selenium. Biological hydrogen catalysts are termed hydrogenases, and though hydrogenases use abundant elements they are extraordinarily complex. This has made it difficult to construct model complexes using inorganic synthesis that can replicate the activities of their biological counterparts. One way to circumvent this problem is to use microbial hydrogen production and let microbes produce and maintain these enzymes inside a cell. Microbial hydrogen production also has the added benefit that hydrogen production could be engineered to connect with other metabolic processes such as photosynthesis and fermentation. Engineering microbes for hydrogen production could eventually allow for the production of hydrogen using inexpensive energy inputs such as solar energy or waste materials. Yet, there are many barriers that need to be overcome in order to engineer a robust microbial organism. One of the primary difficulties of developing this technology has been the oxygen sensitivity of hydrogenases. Hydrogenases when exposed to atmospheric concentrations of oxygen either completely inactivate or their rates are significantly slowed. To engineer a hydrogenase that is more amenable for microbial hydrogen production, the optimization of expressing and purifying hydrogenase enzymes has been developed. Methodologies have been developed to characterize how oxygen inactivates hydrogenase enzymes, and a new methodology has been explored to help find novel hydrogenase gene sequences that may help in engineering oxygen tolerant enzymes.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 Biochemical, spectroscopic, and structural investigations on [FeFe]-hydrogenase maturation and complex metallocluster assembly(Montana State University - Bozeman, College of Letters & Science, 2010) Mulder, David Wayne; Chairperson, Graduate Committee: John W. PetersMetals are present in nearly half of all enzymes, often at the active site, where they modulate catalytic function. Some of these metalloenzymes exist with a single bound metal ion while many others contain complex metal clusters. Complex FeS assemblies are associated with the interconversion of the small molecules H 2, CO, CO 2, N 2, and NH 3. One such complex metalloenzyme, [FeFe]-hydrogenase, catalyzes the reversible oxidation of molecular H 2. The active site of [FeFe]-hydrogenases, the Hcluster, exists as a [4Fe-4S]-subcluster bridged by a protein thiolate ligand to a 2Fesubcluster which contains biologically unique CO and CN- ligands and a dithiolate ligand. The H-cluster is synthesized by the activities of the hydrogenase maturation enzymes HydE, HydF, and HydG and until recently little was known concerning the biosynthetic pathway for the H-cluster. The results presented here provide significant insight into the stepwise mechanism of H-cluster biosynthesis. Biochemical and spectroscopic characterization of the structural [FeFe]-hydrogenase enzyme expressed in a genetic background devoid of maturation genes hydE, hydF, and hydG (HydA Delta EFG) indicates by the presence of a [4Fe-4S] cluster required for [FeFe]-hydrogenase activation that the [4Fe-4S]-subcluster and 2Fe-subcluster of the H-cluster are synthesized independently. The determination of the x-ray crystal structure of HydA Delta EFG confirms this by revealing the presence of the [4Fe-4S]-subcluster and an open binding pocket for the 2Fe-subcluster, indicating that H-cluster synthesis is directed in a stepwise manner with synthesis and insertion of the [4Fe-4S]-subcluster occurring first by generalized host cell machinery followed by synthesis and insertion of the 2Fe-subcluster by specialized hyd encoded maturation machinery. The structure also reveals that insertion of the 2Fe-subcluster occurs through a positively charged channel that collapses following incorporation, as a result of conformational changes in two conserved loop regions. By utilizing complementary gene data base searching with these structural studies, new insight is made known into the evolutionarily relationships between [FeFe]-hydrogenases present in microorganisms and the eukaryotic Nar1 family of proteins which function in iron-sulfur cluster biosynthesis. The work presented as a whole, by establishing parallels to complex metal cofactor biosynthesis in nitrogenase, reveals unifying themes in complex metal cluster assembly and fundamental features of metalloenzyme evolution.Item Investigations on [FeFe]-hydrogenase active site biosynthesis(Montana State University - Bozeman, College of Letters & Science, 2010) McGlynn, Shawn Erin; Chairperson, Graduate Committee: John W. PetersHydrogenase enzymes, which catalyze the reversible oxidation of molecular hydrogen, occupy important roles as catalysts in microbial energy transfer and conservation. This seemingly simple reaction between protons and electrons necessitates the utilization of some of nature's most complicated organo-metallic cofactors. Remarkably, two evolutionarily independent types of enzymes capable of catalyzing this reaction exist - termed the [NiFe] and [FeFe]-hydrogenases. The biosynthesis of the cofactors harbored by these enzymes poses questions as to the assembly pathways involved in constructing hydrogen competent catalysts, and herein research as to the biosynthesis of the [FeFe]-hydrogenase active site is presented. Data as to the protein components involved in this process are presented which include the development of an E.coli based expression system for hydrogenase maturation protein factors, their isolation, and the first functional assignment of two of these proteins. The HydF protein is shown to be operative as an H-cluster intermediate bearing scaffold for [FeFe]-hydrogenase active site assembly, and the HydG protein is demonstrated to be responsible for the formation of cyanide and carbon monoxide from tyrosine. In addition, observations of a novel radical SAM enzyme is reported in conjunction with its putative involvement in the biosynthesis of the Hmd-hydrogenase found in methanogens. Together, these observations contribute to understanding biology's ability to construct complex organo-metallic cofactors, and lay a foundation for the consideration of the evolutionary events that led to the biological ability to assemble complex metallocofactors.Item Multi-edge X-ray absorption spectroscopy and electronic structure calculations of biomimetic model complexes of the H-cluster of [FeFe]-hydrogenase(Montana State University - Bozeman, College of Letters & Science, 2012) Giles, Logan James; Chairperson, Graduate Committee: Robert K. Szilagyi; Alexios Grigoropoulos and Robert K. Szilagyi were co-authors of the article, 'Multi-edge x-ray absorption spectroscopy part I: Xanes analysis of a biomimetic model complex of [FeFe]-hydrogenase' in the journal 'Journal of physical chemistry B' which is contained within this thesis.; Alexios Grigoropoulos and Robert K. Szilagyi were co-authors of the article 'Electron and spin density topology of the H-cluster and its biomimetic complexes' in the journal 'European journal of inorganic chemistry' which is contained within this thesis.FeFe-hydrogenases are members of a family of metalloenzymes that catalyze the conversion of protons and electrons to dihydrogen at a remarkable rate. The catalytic center of this enzyme, the H-cluster, contains a classical [4Fe-4S] cluster that is covalently and magnetically coupled through a cysteine residue to a 2Fe-subcluster. The 2Fe-subcluster contains normally biotoxic carbonyl and cyanide ligands and a dithiolate ligand that is unique in biology. Many biomimetic model complexes have been synthesized that attempted to mimic the H-cluster reactivity, but none have been successful at as low of a reduction potential and as high of a reaction rate as the metalloenzyme. Thus the goal of this research is to develop a blueprint for understanding the electronic structure of the H-cluster, through functionally analogous model complexes. The first step towards this goal is to carry out multi-edge X-ray absorption spectroscopic measurements and electronic structure calculations. We first developed the multi-edge X-ray absorption spectroscopy method for a prototypical biomimetic complex, Fe 2(u-S(CH 2) 3S)(CO) 6. This allowed for the complete definition of the orbital composition for the unoccupied frontier orbitals. We used this information to calibrate our computational results in order to accurately describe similar biomimetic model complexes. We used the multi-edge X-ray absorption spectroscopic approach and the calibrated computational models to analyze four structural features of the 2Fe-subcluster of the H-cluster through representative biomimetic model complexes. We find unique trends for each series that helped to develop an understanding of how each compositional feature contribute to structure. These insights can be used for optimizing model complexes with potential to match the reactivity of the FeFe-hydrogenase enzymes. We also used our calibrated electronic structure method to analyze the spin density at the bridgehead position of the unique dithiolate ligand and dissect the intricate details of the electronic structure for the protein-environment embedded H-cluster model.