Nitric oxide (NO) is a small, gaseous molecule that is toxic to life at high doses but serves a crucial role in biological processes at lower concentrations, including: cell signaling, immune response, and more recently, as a synthon in the biosynthesis of natural products in bacteria. Metalloenzymes are incredibly versatile catalysts that enable chemistry that often, still has no comparable laboratory reaction.
TxtE, a cytochrome P450 (CYP), utilizes NO as a co-substrate along with dioxygen (O2) to catalyze the regioselective nitration of L-tryptophan (Trp) to produce 4-NO2-Trp. Work in this dissertation established that the TxtE ferric-superoxo intermediate is resistant to reduction, which facilitates its reaction with diffusible NO en route to an , as yet,-uncharacterized nitrating species. Furthermore, it is shown that an outer-sphere protein residue influences the nitration chemistry of TxtE. A Thr250Ala mutant version of TxtE characterized and found to lack all nitration ability despite maintaining cofactor incorporation and retaining competence for formation of the ferric-superoxo intermediate. Separately, experiments performed with wild-type TxtE demonstrate that analogs of Trp affect the lifetime of the ferric-superoxo intermediate and enable substrate hydroxylation.
Additionally, a non-heme, diiron enzyme from Mycobacterium kansasii (MkaHLP) was previously established to possess NO peroxidase activity. In this dissertation, a Tyr54Phe mutant form of MkaHLP was characterized and found to have greatly diminished NO peroxidase activity due to the removal of the characteristic tyrosine ligand to the diiron site. Implications of this change in activity are discussed in the relevant section.
| Identifer | oai:union.ndltd.org:ucf.edu/oai:stars.library.ucf.edu:etd2023-1418 |
| Date | 01 January 2024 |
| Creators | Martin, Christopher P. |
| Publisher | STARS |
| Source Sets | University of Central Florida |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | Graduate Thesis and Dissertation 2023-2024 |
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