Date of Award
6-21-2024
Degree Type
Dissertation
Degree Name
Doctor of Philosophy (PhD)
Department
Chemistry
First Advisor
Graham Moran
Abstract
Flavin is a molecule composed of a tricyclic isoalloxazine ring linked to a ribityl. Flavins are utilized as a cofactor by many enzymes to facilitate a myriad of chemistries. Flavoproteins were first discovered in the late 1800 s and have since been an interest to researchers, largely due to the unique spectrophotometric characteristics that make it a valuable reporter molecule. Flavin cofactors (typically either flavin adenine dinucleotide (FAD) or flavin mononucleotide (FMN)) are chemically versatile as the isoalloxazine ring can form one and two-electron reduced states. The reduced states are prone to react with molecular oxygen and numerous observations have been compromised by their inclusion of dioxygen. For this reason, my research has focused on the investigation of several poorly understood/enigmatic flavoproteins employing stringent anaerobic technique. Transient state conditions were utilized to isolate various chemical intermediates and discern individual steps of an enzyme’s catalytic mechanism. Specifically, my research has most recently focused on the study of the FAD-containing enzyme, thioredoxin glutathione reductase (TGR), found within parasitic flatworms of the genus Schistosoma. This enzyme utilizes a relay of disulfide pairs to transport electrons from NADPH to reduce either thioredoxin or glutathione, maintaining the organism’s redox homeostasis. Analysis of this enzyme has revealed a unique electron distribution pathway consisting of various quasi-equilibrium states. Additionally, I have expanded our lab’s investigation of mammalian dihydropyrimidine dehydrogenase (DPD), which consists of an extensive protein scaffold encapsulating an electron conduit composed of two FADs, eight iron-sulfur centers, and two FMNs, to catalyze simple hydride transfer chemistry. My work focused on utilizing a charge transfer transition to answer some of the outstanding questions that persisted throughout much of our laboratories previous analysis of DPD. Although not flavin containing enzymes, I also characterized the catalytic mechanism of E. coli GTP cyclohydrolase II (RibA) and RibD, both of which are involved in the synthesis of the FAD/FMN precursor, riboflavin.
Recommended Citation
Smith, Madison M., "Enzymological Revisionism: Employing Transient State Approaches to Define the Catalytic Sequences of Poorly Understood Systems" (2024). Dissertations. 4078.
https://ecommons.luc.edu/luc_diss/4078
