Theses and Dissertations at Montana State University (MSU)
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Item How biological catalysts activate oxygen to realize its full potential(Montana State University - Bozeman, College of Letters & Science, 2021) Ellis, Emerald Sue; Chairperson, Graduate Committee: Jennifer DuBois; Daniel J. Hinchen, Alissa Bleem, Lintao Bu, Bennett R. Streit, Quinlan V. Doolin, William E. Michener and Brandon C. Knott were authors and Sam J.B. Mallinson, Mark D. Allen, Melodie M. Machovina, Christopher W. Johnson, Gregg T. Beckham, John E. McGeehan, Jennifer L. DuBois were co-authors of the article, 'Engineering a biocatalyst for demethylation of lignin-derived aromatic aldehydes' in the journal 'Journal of the American Chemical Society Au' which is contained within this dissertation.; Luke MacHale, Robert K. Szilagyi and Jennifer L. DuBois were co-authors of the article, 'How chemical environment activates anthralin and molecular oxygen for direct reaction' in the journal 'Journal of organic chemistry' which is contained within this dissertation.; Dissertation contains an article of which Emerald Sue Ellis is not the main author.Dioxygen is a potent oxidant, inexpensive, and environmentally-friendly compared with most industrial oxidants, but intrinsic energy barriers to reaction limit its utility. Biological catalysts can activate O 2 by generating dangerous reactive oxygen species intermediates. The fundamental chemistry of two diverse O 2-utilizing enzyme systems were examined: GcoAB, a cytochrome P450 which catalyzes the O-demethylation of aromatic alcohols using heme to activate O 2, and NMO, an antibiotic biosynthesis monooxygenase which catalyzes cofactor-independent monooxygenation of an organic substrate. The enzyme active site environments and the reactions catalyzed therein were investigated with mutagenesis, X-ray crystallography, molecular dynamics simulations, fluorescence and UV/visible spectroscopy, cyclic voltammetry, electrode-based measurement of O 2 consumption, high-performance liquid chromatography, and simulations of homogenous solvation using quantum chemistry composite methods. The substrate range of GcoAB was expanded by rational design engineering to include two aromatic aldehydes commonly found in chemically-processed lignin. Only a single-point mutation was needed for GcoAB to catalyze demethylation of each new substrate. The reaction catalyzed by NMO can be called 'substrate-assisted' because the substrate mimics the role of the organic cofactor flavin in activating O 2. The physics of this reaction were probed using Marcus Theory, which relates the activation energy of the reaction to the free energy and the reorganization energy. By measuring the differences in the activation energy and free energy of the reaction within and without the enzyme, we found that the enzyme mainly acts on the reorganization energy term. The reaction was then examined in several homogenous solvents chosen based on their chemical similarity to individual amino acids. Homogenous solvation is much less computationally expensive to model than a protein active site, especially at higher levels of theory. By this approach, we discovered a plausible mechanism by which the chemical environment alone can boost the O 2-activating capacity of NMO's substrate--particularly by stabilizing the deprotonated anion which can transfer an electron to O 2 more easily than the neutral molecule. In summary, this work demonstrates that, while cofactors are responsible for activating O 2 in most oxidases, full appreciation of how an oxidase catalyzes reactions requires that neither the enzyme environment nor the substrate be ignored.Item Evaluation of mixed-oxidants against sodium hypochlorite for the disinfection and removal of biofilms from distribution systems(Montana State University - Bozeman, College of Engineering, 1997) Crayton, Cynthia Lynn; Chairperson, Graduate Committee: Anne CamperProblem Statement: As drinking water regulations are applied to smaller utilities, an area of emerging concern for the water industry is the installation of disinfection systems to meet the newly imposed standards. Since traditional disinfection technologies are usually beyond the safety, economic, and/or site restraint considerations for small systems, an alternative is required. The mixed-oxidants disinfection system (MIOX) appears to provide a reasonable alternative for small distribution systems as a safe, reliable, and cost effective technology that is easy to operate and is readily compatible with other treatment systems. The goal of this five-phase study was to evaluate the potential of the MIOX disinfectant (produced on-site using feedstocks of ordinary salt, water, and twelve volt electricity) against free chlorine for biocidal efficacy, biofilm/biofouling removal, biofilm regrowth potential, relative corrosion potential, and cost effectiveness. Although mixed-oxidants have been proven effective in potable water disinfection, biofilm removal is a new application for this alternative disinfection technology.Item Chemical approaches to probe environmental stress in Archaea(Montana State University - Bozeman, College of Letters & Science, 2009) Tarlykov, Pavel Victorovich; Chairperson, Graduate Committee: Brian BothnerLittle is known about strategies and mechanisms employed by thermophilic organisms to adapt to environmental stress. Sulfolobus solfataricus is a thermophile that belongs to Archaea, the third domain of life, and can be found in unusual habitats, such as the hot springs of Yellowstone National Park. This archaeon can tolerate high temperature, extreme acidity and high concentrations of heavy metals and other toxic substances. Thus, S. solfataricus has been chosen by many researchers as a model system for biochemical, structural, and genetic studies. In this work S. solfataricus has been exposed to hydrogen peroxide as a natural mild oxidant and arsenic as a common toxic metalloid. One of the aims was to quantitatively define the regulation of proteins upon treatment with hydrogen peroxide or arsenic species in different time periods and concentrations. In this sense, two-dimensional gel electrophoresis approach in conjunction with novel chemical tagging probes has been applied to detect changes on the level of regulation and chemical modification of individual proteins within the whole proteome in response to the stressors. Proteins expression levels have been monitored, redox-sensitive and phosphoproteomic profiles of the S. solfataricus proteome have been identified. Synthesis of the results has allowed a general scheme for how S. solfataricus fights H₂O₂- and As-induced stress. Lists of mapped proteins have been created and potential biomarkers for oxidative stress have been identified, which can guide further research to better understand mechanisms of proteomic response to the environmental stress in Archaea on the example of thermophilic archaeon S. solfataricus.