Theses and Dissertations at Montana State University (MSU)
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Item Delineating the determinants of carboxylation in 2-ketopropyl coenzyme M oxidoreductase/carboxylase: a unique CO 2-fixing flavoenzyme(Montana State University - Bozeman, College of Letters & Science, 2018) Prussia, Gregory Andrew; Chairperson, Graduate Committee: John W. Peters; George H. Gauss, Florence Mus, Leah Conner, Jennifer L. DuBois and John W. Peters were co-authors of the article, 'Substitution of a conserved catalytic dyad causes loss of carboxylation in 2-KPCC' in the journal 'Federation of European Biochemical Societies letters' which is contained within this dissertation.; Jennifer L. DuBois and John W. Peters were co-authors of the article, 'A role for hisitidine 506 in carboxylate stabilization of 2-ketopropyl coenyzme M oxidoreductase/carboxylase' which is contained within this dissertation.; Gregory Andrew Prussia is not the main author of an article which is contained in this dissertation.Global CO 2-emissions are continuously rising, accelerating the impact of associated environmental processes such as climate change, deforestation, and ocean acidification. As a consequence, there is great interest in processes that can mitigate the increase in anthropogenic CO 2. The biological incorporation of a CO 2 molecule into an organic substrate is catalyzed by enzymes known as carboxylases. Although carboxylases employ diverse CO 2-fixing mechanisms and play broad physiological roles in Nature, they follow three general paradigms: 1). The formation of a reactive ene-intermediate nucleophile. 2). Protection of this reactive nucleophile from potential competing electrophiles (other than CO 2) by excluding solvent from the active site. 3). Electrostatic complementation of the negatively-charged carboxylation intermediate and product. 2-ketopropyl coenzyme M oxidoredutase/carboxylase (2-KPCC) is the only known carboxylating member of the FAD-containing, NAD(P)H-dependent disulfide oxidoreductase (DSOR) enzymes. The members of this family catalyze redox reactions and several well-characterized members catalyze the reductive cleavage of disulfide substrate. 2-KPCC performs the reductive cleavage of a thioether bond and subsequently carboxylates it's intermediate. How 2-KPCC has integrated the paradigms of carboxylation using a scaffold purposed for reductive cleavage is unknown. In this work, the paradigms mentioned above are identified in 2-KPCC and the methods by which 2-KPCC integrates carboxylation chemistry with reductive cleavage are discussed. Essential to the redox chemistry catalyzed by many DSOR members is a conserved His-Glu catalytic dyad, which serves to stabilize the electronic interaction between the FAD cofactor and the redox-active cysteine pair in the reactive state. 2-KPCC has substituted the catalytic His and Glu with Phe and His, respectively. We show that the Phe substitution is critical for excluding protons (as competing electrophiles) from the active site and the downstream His substitution acts to stabilize the negative charge on the carboxylated product, acetoacetate. Individually, each substitution plays an essential role in carboxylation. We show through a detailed spectroscopic study that by substituting both catalytic dyad residues the protonated and electronic state of the redox-active cysteine pair and FAD cofactor are affected, altering the DSOR active site to accommodate the unique cleavage and CO 2-fixation reaction catalyzed by 2-KPCC. Thus, this research has furthered the understanding of how the prototypical reductive cleavage reactions catalyzed by DSOR enzymes can be coordinated with a carboxylation reaction by a mechanism analogous to that shared by established carboxylase enzymes.Item Partitioning of reactive oxygen species via the re-oxidation of electron transfer flavoprotein(Montana State University - Bozeman, College of Letters & Science, 2019) Austvold, Chase Kennor; Chairperson, Graduate Committee: Edward DratzThe biology of Reactive Oxygen Species are poorly understood. Within a healthy cell, Reactive Oxygen Species behave as signaling molecules, although overproduction leads to oxidative damage. In order to understand when the overproduction of Reactive Oxygen Species takes place, or leads to oxidative damage, the elementary step of quantification becomes necessary. Electron Transfer Flavoprotein is a known Reactive Oxygen Species producing enzyme and was studied. Electron Transfer Flavoprotein is a key-player within the production of energy within the eukaryotic mitochondria. The redox nature of Electron Transfer Flavoprotein's catalytic cofactor, flavin adenine dinucleotide produced two types of ROS; the superoxide anion (O 2 °-) and hydrogen peroxide (H 2 O 2). Electron Transfer Flavoprotein produced roughly five-fold more O 2 °-compared to H 2 O 2 as the enzyme became oxidized. It has been put forward that the production of these two Reactive Oxygen Species is dictated by the formation of a radical pair between the flavin adenine dinucleotide of Electron Transfer Flavoprotein and molecular oxygen. Two types of radical pairs can be formed, either in a triplet or singlet state, and the rate in which these states occur can be influenced by a static magnetic field. Therefore, the effect of a magnetic field on these products was also studied. Upon the suppression of magnetic field strength, the production of H 2 O 2 decreased and a proportional increase of O 2 °-was observed.Item Evolution and function of flavin-based electron bifurcation(Montana State University - Bozeman, College of Letters & Science, 2018) Poudel, Saroj; Chairperson, Graduate Committee: Eric Boyd; John W. Peters (co-chair); Eric C. Dunham, Melody R. Lindsay, Maximiliano J. Amenabar, Elizabeth M. Fones, Daniel R. Colman and Eric S. Boyd were co-authors of the article, 'Origin and evolution of flavin-based electron bifurcating enzymes' in the journal 'Frontiers in microbiology' which is contained within this thesis.; Amaya M. Garcia Costas was an author and Anne-Frances Miller, Gerrit J. Schut, Rhesa N. Ledbetter, Kathryn R. Fixen, Lance C. Seefeldt, Michael W. W. Adams, Caroline S. Harwood, Eric S. Boyd and John W. Peters were co-authors of the article, 'Defining electron bifurcation in the electron transferring flavoprotein family' in the journal 'Journal of bacteriology' which is contained within this thesis.; Daniel R. Colman was an author and Kathryn R. Fixen, Rhesa N. Ledbetter, Yanning Zheng, Natasha Pence, Lance C. Seefeldt, John W. Peters, Caroline S. Harwood and Eric S. Boyd, were co-authors of the article, 'Electron transfer to nitrogenase in different genomic and metabolic backgrouns' in the journal 'Journal of bacteriology' which is contained within this thesis.Anaerobic microorganisms live in energy limited environments with low nutrient fluxes. Thus, selection has likely acted on these cells to innovate mechanisms that improve the efficiency of anaerobic energy metabolism. In 2008, the process of flavin-based electron bifurcation (FBEB) was discovered and has since been shown to be a critical process that allows anaerobic cells to overcome thermodynamic barriers and to improve metabolic efficiency. FBEB enzymes catalyze the coupling of exergonic and endergonic oxidation--reduction reactions with the same electron donor to circumvent thermodynamic barriers and minimize free energy loss. To date, a total of 12 FBEB enzymes have been discovered that share common features that include the presence of protein-bound flavin, the proposed site of bifurcation, and the electron carrier ferredoxin. Due to its recent discovery, a comprehensive description of the natural history of bifurcating enzymes is lacking. In this thesis, we report the taxonomic and ecological distribution, functional diversity, and evolutionary history of bifurcating enzyme homologs in available complete genomes and environmental metagenomes. Moreover, we investigated the functional and ecological constraints that led to the emergence of FBEB enzymes. Bioinformatics analyses revealed that FBEB enzyme homologs were primarily detected in the genomes of anaerobes, including those of sulfate-reducers, acetogens, fermenters, and methanogens. Phylogenetic analyses of these enzyme homologs suggest that they were not a property of the Last Universal Common Ancestor of Archaea and Bacteria indicating that they are a more recent evolutionary innovation. Consistent with the role of these enzymes in the energy metabolism of anaerobes, FBEB homologs were enriched in metagenomes from subsurface environments relative to those from surface environments. In fact, the earliest evolving homologs of most bifurcating enzymes were detected in subsurface environments, including fluids from subsurface rock fractures and hydrothermal systems. Together, these data highlight the central role that FBEB played and continued to play in the energy metabolism of anaerobic microbial cells inhabiting subsurface environments.Item Quantitative prediction of dye fluorescence quantum yields in proteins(Montana State University - Bozeman, College of Letters & Science, 2009) Hutcheson, Ryan Mitchell; Chairperson, Graduate Committee: Patrik R. CallisThe application of a method previously developed by Callis et al. to predict the quantum yields of Trp fluorescence has been successfully applied to the fluorescence of fluorescein and flavins in proteins. The calculated lifetime range of 2 ps - 4 ns is in agreement with experiment. The fluctuations in the electron transfer rate are shown to be dictated by the fluctuations in the density of states. This is evident by the comparison of the fractional deviation of the interaction, density of states and the rate. Here the fluctuations in the density of states is an order of magnitude larger than the fluctuations in the interactions and is nearly the same as that of the kET fluctuations. This demonstrates that the fluorescence lifetime variability is controlled by the electrostatic environment and not the distance dependence of the interaction.