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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.