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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 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 Oxidation of human nitrosylhemoglobin monitored by UV-Vis and EPR spectroscopies : detection of products and intermediates(Montana State University - Bozeman, College of Letters & Science, 2005) Williams, Elizabeth Mary; Chairperson, Graduate Committee: David J. SingelOnce viewed only as a toxic free radical, nitric oxide (NO) has been established as an essential and ubiquitous signaling and regulatory molecule in biological systems. Notably, NO was identified as the endothelium-derived relaxing factor (EDRF) in the blood. NO is capable of complex redox chemistry and interaction with a host of protein families. Among these proteins is hemoglobin (Hb) which can interact with NO at the level of the heme and can bind NO at Cys93 on its â subunit to form S-nitrosylated Hb (SNO-Hb). NO bound as SNO-Hb is chemically labile and thus preserves bioavailability of NO. However, when NO reacts with oxyHb or deoxyHb NO bioavailability is quenched by conversion to nitrate or by tightly binding the heme, respectively. Therefore, the question is raised as to how NO can be EDRF in the presence of such high Hb concentrations in the blood. One way NO availability can be preserved is by exploiting the redox chemistries of both Hb and NO. Human Hb(NO)4 oxidation by K3Fe(CN)6 was studied and products and intermediates were identified by UV-Vis and EPR spectroscopies. Periodically, samples were withdrawn from the reaction mixture for nitrosylation product and/or EPR analysis. Reaction spectra converted to heme species concentration vs. time plots through leastsquares fitting of five basis spectra. These data were then utilized to generate a de minimis model of the oxidation reaction. We demonstrate that the oxidation of Hb(NO)4 by K3Fe(CN)6 1) leads to the production of SNO-Hb, 2) occurs preferentially at the â heme, and 3) proceeds through an HbFeIIINO intermediate.Item Spin-label electron paramagnetic resonance investigations of PAMAM dendrimer end-group structure and dynamics(Montana State University - Bozeman, College of Letters & Science, 2007) Sebby, Karl Bernell; Chairperson, Graduate Committee: David J. Singel; Patrik Callis (co-chair)PAMAM dendrimers are nanoparticles containing a series of branching units emanating from an ethylene diamine initiator core. Control of the number of branching units during synthesis results in monodisperse macromolecules with a specified, but variable, number of terminal branches, to which various functionalities can be attached. The ability to attach large numbers of functional groups, in controlled ratios, to the dendrimer end-groups makes dendrimers attractive templates for a variety of applications. For example, partially glycosylated dendrimers are being explored as multivalent ligands for inhibitory and targeting purposes. In such applications the spatial distribution of functional groups on the dendrimers must be understood. Analytical studies aimed at elucidating the structure and dynamics of dendrimers have, to date, been very limited. In this dissertation, a spin-label electron paramagnetic resonance (EPR) approach is developed and applied to solve this problem.Item Spin-label electron paramagnetic resonance characterization of heterogeneously functionalized dendrimers(Montana State University - Bozeman, College of Letters & Science, 2004) Walter, Eric Dale; Chairperson, Graduate Committee: David Singel; David Dooley (co-chair)Dendrimers are roughly spherical macromolecular polymers that can provide a molecular framework for the attachment of various functionalities. The range of application can be vastly expanded when attached with a heterogeneous mix of functionalities tailored to meet a specific need. The effective application requires the acquisition of several new techniques. First, chemical control of the average ratio of functional groups and analytical methods to determine the ratio. Secondly, a determination of the distribution of ratios. And finally, a method whereby the spatial distribution is determined. The ratio and distribution of ratios were determined by MALDI-TOF Mass Spectrometry. The average ratio was easily controlled by using stoichiometric ratios of reactants and the distribution of ratios was found to follow a normal distribution. This was determined by the analysis of the MALDI spectral linewidths. The spatial distribution was determined by a Spin-Label Electron Paramagnetic Resonance technique. By adapting and combining mathematical and spectroscopic techniques from other related systems, it was found that the EPR spectrum was sensitive to the spatial distribution of spin-labels included as one of the attached functional groups. The dipolar interaction between spins lead to an increase in spectral line-broadening that could be compared to computer simulations of different spatial distributions. For all cases studied the distribution was found to be random.