Date of Award

Summer 2017

Document Type


Degree Name

Doctor of Philosophy (PhD)



First Advisor

Kincaid, James R.

Second Advisor

Dockendorff, Christopher

Third Advisor

Timerghazin, Qadir


Cytochrome P450 is a broad class of heme monooxygenase enzymes which catalyze various oxidative transformations. There are two main kinds of mammalian P450s: steroidogenic and drug metabolizing P450s. The first project involves a steroidogenic P450, CYP17A1, occupying a central role in the biosynthesis of steroid hormones. It catalyzes hydroxylation reaction on pregnenolone and progesterone, generating 17OH-pregnenolone and 17OH-progesterone, presumably utilizing a “Compound I” species. However, these hydroxylated products can be further processed in a second oxidative cycle to cleave the C17–C20 bond to form dehydroepiandrosterone or androstenedione, respectively, a crucial step in androgen production. Interestingly, it is well known that cytochrome b5 is a key regulator of androgen synthesis, by a mechanism that is still not well understood. As the enzymes involved here are both membrane-bound, resonance Raman (rR) studies are performed on unique nanodisc-based dyads to investigate their interaction, including characterization of their ferric state and unstable dioxygen intermediates, the essential results supporting an electron transfer role of cyt b5. The second project is focused on a fusion enzyme called P450BM3, which possesses a covalently linked cytochrome P450 reductase. This fusion enzyme is clearly a good target for biotechnologically application. In the present work, rR studies focus on several biotechnologically important mutants of P450BM3, which have altered substrate selectivity, allowing metabolism of the human proton pump inhibitor, omeprazole, producing metabolites that precisely match those generated by human CYP2C19, thereby providing an inexpensive source of these precious materials that are needed for drug metabolism studies. Specifically, it is shown that binding of omeprazole to these BM3 mutants leads to differing degrees of high spin state conversion of the heme iron. Furthermore, the ferrous CO adduct were acquired to interrogate the effect of substrate binding on the distal pocket architecture. The third project involves the biotechnologically important peroxygenases. Specifically, rR studies are applied to CYP152L1 and CYP152L2 enzymes, which are able to convert fatty acids into industrially valuable terminal alkenes without requiring a complex reductase system. Herein, using chemical strategies to trap and isolate key reaction intermediates, rR spectroscopy is being employed to probe their structure and reactivity.