Date of Award

2026

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Chemistry

First Advisor

Graham Moran

Abstract

Flavins are ubiquitous redox active cofactors that can catalyze electron transfer between substrates. They offer the advantage of being able to stabilize a range of oxidation and protonation states, as well being amenable to covalent modification, which can further alter their activity. As such, the scope of reactions catalyzed by flavoproteins is broad and flavin has implications in a range of biological processes. The two predominant forms of flavin that serve as redox active cofactors are flavin adenine mononucleotide (FMN) and flavin adenine dinucleotide (FAD), which are functionally homologous. A major complication of studying flavoprotein dehydrogenases, and one that has often been overlooked, is the propensity of reduced flavins to react with molecular oxygen. As such, valid measurements typically require strict anaerobic conditions. Here, a transient-state kinetic approach is utilized under strict anaerobic conditions to elucidate mechanistic details about several complex flavoprotein dehydrogenases. Thioredoxin/glutathione reductase (TGR) from Schistosoma mansoni is a member of the flavin disulfide reductase family of enzymes and catalyzes the reduction of thioredoxin and glutathione using electrons derived from NADPH. It is the focal point of ROS detoxification in schistosomes, and as such has been identified as a potential drug target. This work has clarified the movement of electrons through the enzyme, which contains an FAD and three redox active disulfides. The site of glutathione reduction has also been unambiguously identified as being at the disulfide most proximal to the flavin, contrary to prior assertions that it was in the N-terminal domain. Dihydropyrimidine dehydrogenase (DPD) from Escherichia coli was also studied, and a mechanism was elucidated that is remarkably similar to that of the mammalian enzyme, with some notable differences. It was found that the enzyme undergoes reductive activation and displays effector roles for both the reductive and oxidative substrates, much like the mammalian enzyme. However, unlike the mammalian enzyme, EcDPD reductively activates to the FADH2 state of the enzyme (rather than the FMNH2 state) and traversal of the first electron from the FAD to the FMN, via the iron-sulfur centers, was found to be rate limiting in turnover. Ferroptosis suppressor protein 1 (FSP1) is a quinone/NAD(P)H oxidoreductase that relies on a single flavin cofactor. It suppresses ferroptosis by generating quinols that intercept ROSs, preventing lipid peroxidation and aiding in neoplastic cell survival. As such, it has been identified as a drug target for a variety of cancers. An anaerobic transient-state analysis has been employed in conjunction with a variety of other methods to elucidate the mechanism and contextualize the physiological role of FSP1. It was found that FSP1 binds NADP+ tightly and release of this tightly bound product is rate limiting in turnover. Quinone reduction is very rapid, resulting in ostensibly no buildup of the reduced state of the enzyme, preventing oxygen reduction or adventitious hydroxylation of the cofactor, despite the pervasive reports of 6-OH- FAD being the active cofactor of FSP1.

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