Biochemical, spectroscopic, and computational studies of metalloenzyme structure and mechanism

Jodts, Richard Jacob

doi:https://doi.org/10.21985/n2-mcxs-7144

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Biochemical, spectroscopic, and computational studies of metalloenzyme structure and mechanism

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Metalloenzymes catalyze remarkable reactions in Nature using transition metal ions. Common earth-abundant metals like copper, iron, zinc, and magnesium catalyze reactions that are the basis of life. These metal sites lend their chemistries to facilitate these reactions, making studying the structure and properties important in understanding the enzymes' abilities and the greater role in the life processes. This thesis combines techniques of biochemistry, inorganic spectroscopy, and computational chemistry to understand three different metalloenzyme active sites and mechanism. The first part of this thesis investigates Nature’s predominate enzyme for methane oxidation, particulate methane monooxygenase(pMMO). Specifically, it investigates on an atomic level the copper centers that perform this oxidation. These chapters highlight the importance of parallel structural and spectroscopy experiments to understanding metal sites within biology. These two techniques complement each other and allowed for more in-depth assignments and characterization of the copper centers. Importantly, within this thesis is the first evidence of a copper center in pMMO interacting with a hydrocarbon-based molecule, a product analogue. This finding identifies one of the copper sites as the site of methane oxidation, representing major progress in understanding the chemical mechanism of pMMO and methane oxidation in Nature. This thesis also studies a diiron enzyme cofactor within an enzyme involved in a natural product biosynthesis, MbnBC. These chapters highlight again the need for parallel methods and approaches to better understand enzyme mechanism. Using parallel spectroscopy and biochemical activity assays, the active state of the catalytic cofactor was elucidated. Structural and additional spectroscopic studies were then used to determine the coordination environments of the catalytic iron ions as well as how substrate binds. Lastly, a combination of biochemistry, spectroscopy, and computational methods was employed to understand the universal intermediates of one of the largest and most important superfamilies of metalloenzymes, the radical SAM enzymes. This section presents some of the first applications of computational methods, as well as new theory, to accurately characterizing these intermediates. Together these three sections present progress in understanding the structure and mechanism of important metalloenzymes.

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