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Item Investigating the metalloproteome of bacteria and archaea(Montana State University - Bozeman, College of Letters & Science, 2024) Larson, James Daniel; Chairperson, Graduate Committee: Brian Bothner; This is a manuscript style paper that includes co-authored chapters.Metalloproteins are proteins that rely on a bound metal for activity and comprise 30-50% of all proteins which are responsible for catalyzing imperative biological functions. Understanding the interplay between essential and toxic metals in the environment and the metalloproteins from an organism (metalloproteome) is important for a fundamental understanding of biology. A challenge in studying the metalloproteome is that standard proteomic methods disrupt protein-metal interactions, therefore losing information about protein- metal bonds required for metalloprotein function. One of the focuses of my work has been to develop a non-denaturing chromatographic technique that maintains these non-covalent interactions. My approach for investigating the native metalloproteome together with leading- edge mass spectrometry methods was used to characterize microbial responses to evolutionarily relevant environmental perturbations. Arsenic is a pervasive environmental carcinogen in which microorganisms have naturally evolved detoxification mechanisms. Using Escherichia coli strains containing or lacking the arsRBC arsenic detoxification locus, my research demonstrated that exposure to arsenic causes dramatic changes to the distribution of iron, copper, and magnesium. In addition, the native arsRBC operon regulates metal distribution beyond arsenic. Two specific stress responses are described. The first relies on ArsR and leads to differential regulation of TCA-cycle metalloenzymes. The second response is triggered independently of ArsR and increases expression of molybdenum cofactor and ISC [Fe-S] cluster biosynthetic enzymes. This work provides new insights into the metalloprotein response to arsenic and the regulatory role of ArsR and challenges the current understanding of [Fe-S] cluster biosynthesis during stress. Iron is an essential and plentiful metal, yet the most abundant iron mineral on Earth, pyrite (FeS2), was thought to be unavailable to anaerobic microorganisms. It has recently been shown that methanogenic archaea can meet their iron (and sulfur) demands solely from FeS2. This dissertation shows that Methanosarcina barkeri employs different metabolic strategies when grown under FeS2 or Fe(II) and HS- as the sole source of iron and sulfur which changes the native metalloproteome, metalloprotein complex stoichiometry, and [Fe-S] cluster and cysteine biosynthesis strategies. This work advances our understanding of primordial biology and the different mechanisms of iron and sulfur acquisition dictated by environmental sources of iron and sulfur.Item Enzymatic strategies for controlling and harnessing the oxidative power of O 2(Montana State University - Bozeman, College of Letters & Science, 2018) Machovina, Melodie M.; Chairperson, Graduate Committee: Jennifer DuBois; Robert J. Usselman and Jennifer L. DuBois were co-authors of the article, 'Monoxygenase substrates mimic flavin to catalyze cofactorless oxygenations' in the journal 'Journal of biological chemistry' which is contained within this dissertation.; Emerald S. Ellis, Thomas J. Carney, Fikile R. Brushett and Jennifer L. DuBois were co-authors of the article, 'Understanding how a cofactor-free protein environment lowers the barrier to O 2 reactivity' in the journal 'Journal of biological chemistry' which is contained within this dissertation.; Sam J. B. Mallinson, Rodrigo L. Silveira, Marc Garcia-Borras, Nathan Gallup were authors and Christopher W. Johnson, Mark D. Allen, Munir S. Skaf, Michael F. Crowley, Ellen L. Neidle, Kendall N. Houk, Gregg T. Beckham, Jennifer L. DuBois and John E. McGeehan were co-authors of the article, 'A promiscuous cytochrome P450 aromatic O-demethylase for lignin bioconversion' in the journal 'Nature Communications' which is contained within this dissertation.; Sam J.B. Mallinson was an author and Brandon C. Knott, Marc Garcia-Borras, Alexander W. Meyers, Lintao Bu, Japheth Gado, April Oliver, Graham P. Schmidt, J. Hinchen, Michael F. Crowley, Christopher W. Johnson, Ellen L. Neidle, Christina M. Payne, Gregg T. Beckham, Kendall N. Houk, John E. McGeehan and Jennifer L. DuBois were co-authors of the article, 'Enabling microbial syringol conversion through structure-guided protein engineering' submitted to the journal 'PNAS' which is contained within this dissertation.; Dissertation contains one article of which Melodie M. Machovina is not the main author.Dioxygen, one of Nature's most powerful oxidants, is essential for countless biological reactions. To harness this oxidant's power while minimizing toxicity, enzymes evolved to interact with O 2, activate it, and poise it for catalysis with substrates. This dissertation explores how two very different enzyme families, monooxygenases and a new class of cytochrome P450s, utilize this powerful oxidant. Previously, it was thought that cofactors are essential for O 2 activation; however, a subset of O 2-utilizing enzymes that catalyze direct reactions between substrate and O 2 was recently discovered, including nogalamycin monoxygenase (NMO). To probe how the protein environment affects thermodynamic and kinetic barriers of O 2 activation, we used a suite of techniques, including: UV/vis (transient and conventional) and electron paramagnetic resonance spectroscopies, O 2 consumption, high-performance liquid chromatography (HPLC), and cyclic voltammetry. Here, we provide evidence that the NMO mechanism has similar characteristics to that in flavoenzymes; in NMO, the substrate, acting in lieu of flavin, donates an electron to O 2, activating it to superoxide with the protein environment facilitating this by lowering the reorganization energy. The last half of this dissertation describes the discovery and engineering of a new class of cytochrome P450 enzymes that employ heme-iron oxygen activation to demethylate key lignin degradation products, forming central carbon intermediates that are precursors for bioplastics. The P450 GcoAB, comprised of the oxidase GcoA and the reductase GcoB, is efficient at demethylating G-lignin, but shows poor reactivity towards S-lignin. Using a structure-guided mutagenesis approach, we generated a variant, F169A GcoA, that is more efficient than wild-type at demethylating G-lignin and the only enzyme that efficiently degrades S-lignin. We characterized this variant, and the wildtype enzyme, using biochemical (UV/vis spectroscopy, HPLC), structural (X-ray crystallography), and computational (Molecular Dynamics and Density Functional Theory). Currently, we are testing the in vitro efficiency of additional variants evolved using a directed evolution approach. The results presented in the following chapters explore the mechanisms of several enzymes. Understanding how O2 is activated and utilized across diverse enzymatic systems provides valuable knowledge that can aid in future design and engineering of systems that use this 'green' oxidant, particularly for large-scale industrial applications.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 Biochemical characterization of the six-transmembrane epithelial antigen of the prostate family of metalloreductases(Montana State University - Bozeman, College of Letters & Science, 2015) Kleven, Mark Daniel; Chairperson, Graduate Committee: C. Martin Lawrence; George H. Gauss was the main author, and Mark D. Kleven, Anoop K. Sendamarai, Mark D. Fleming and C. Martin Lawrence were co-authors of the article, 'The crystal structure of six-transmembrane epithelial antigen of the prostate 4 (Steap4), a ferri/cuprireductase, suggests a novel interdomain flavin-binding site' in the journal 'Journal of biological chemistry' which is contained within this thesis.; Mark D. Fleming and C. Martin Lawrence were co-authors of the article, 'Characterization of a single B-type heme, FAD and metal binding sites in the transmembrane domain of six trans-membrane epithelial antigen of the prostate (Steap) family proteins' submitted to the journal 'Journal of biological chemistry' which is contained within this thesis.Iron and copper are the two most abundant transition metals in humans and are mediators of many essential cellular processes. The entry of these metals into cells require controlled processes, including their reduction prior to uptake. A group of integral membrane enzymes, the six-transmembrane epithelial antigen of the prostate (Steap) family, are able to perform this function. Steap3, in particular, functions as the primary ferric reductase in the transferrin cycle, the dominant mode of erythrocyte iron uptake. How these enzymes perform these functions has remained ill-defined. Here, the biochemical underpinnings of Steap metalloreductase activity have been investigated. To elucidate these mechanisms, expression systems for Steap3 and Steap4 have been developed in bacterial, insect, and human cell lines and purified to varying degrees. By analyzing the truncated cytoplasmic oxidoreductase domain of Steap4, it was found that NADPH is oxidized by transferring a pair of electrons to a flavin. With this truncation, however, flavin only binds weakly and the construct shows no ability to preferentially bind one type of flavin. In contrast, when the full length Steap3 was partially purified, it exhibits high-affinity FAD-binding, indicating that the transmembrane region of the protein contains the major structural features of the FAD binding site. Further, it was found that the cytoplasm-oriented loops between transmembrane helices formed the site. The next cofactor in the electron transport chain is a single b-type heme. Two strictly conserved histidines were identified that coordinate the heme and both are required for heme incorporation. The metal binding site at the extracellular face of the membrane was also characterized. Here, it was found that Steap3 and Steap4 share a conserved high-affinity iron binding site. Additionally, iron and copper both bind with similar affinities to Steap4. Two critical residues of the metal binding site were determined and their predicted proximity to the heme cofactor suggests that the electron is transfer is direct between cofactor and metal. Finally, it was found that Steap's are able to dimerize in the cells, forming homo- and heterodimers Together, the enzymatic mechanism has been characterized in-depth for the first time for these physiologically-significant enzymes.Item Expression and characterization of copper-containing proteins: galactose oxidase and tyrosinase(Montana State University - Bozeman, College of Letters & Science, 2002) Kamlin, Ejan MarieItem 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.