Date of Award

Fall 2017

Document Type


Degree Name

Doctor of Philosophy (PhD)



First Advisor

Kincaid, James R.

Second Advisor

Ryan, Michael D.

Third Advisor

Sem, Daniel S.


The Cytochrome P450 (P450s) has been the subject of intense research for over six decades. An efficient approach for isotopic labeling of the prosthetic group in heme proteins was exploited to produce an analogue of the soluble bacterial cytochrome P450cam (P450cam) that contains a 13C labeled-protoheme prosthetic group. HU227 strain of E. coli, which lacks the δ-aminolevulinic acid (δ-ALA) synthase gene, was employed in the heterologous expression of P450cam harboring a prosthetic group labeled with 13C at the Cm and Cα positions by growing cells in the presence of [5-13C] δ-ALA, which was synthesized in four steps from [2-13C] glycine. NMR spectroscopy was used to confirm labelling of the hemes at the Cm and Cα positions. This system was utilized as proof of principle for the strategy of defining active site structure in cytochrome P450cam, including proton-to-proton distances on bound substrates, using NMR methods1. Such data are potentially of significant use in furnishing necessary experimental restrictions in docking routines, which are commonly employed in determining the relative affinities of drug candidates. 2D NOESY was employed and resonances assigned for the 13C labeled reference positions on heme and substrate. To confirm these resonance assignments on camphor, a substrate analogue, norcamphor was used. In another project, though it’s widely accepted that a highly reactive Fe(IV)=O π-cation radical, compound I, facilitates the oxidation of relatively inert hydrocarbons, spectroscopic characterization of this putative intermediate has eluded detection under turnover conditions, presumably due to its very short lifetime. Chemically inert substrates of P450s have been utilized in a novel approach to capture and stabilize this transient intermediate and characterize it with resonance Raman (RR) spectroscopy coupled with cryoradiolysis studies. Specifically, perfluorodecanoic acid was utilized as an inert surrogate substrate of a thermophilic cytochrome P450 designated CYP119 which was reported to possess a stable compound 12. Clearly, the presence of an inert substrate at low temperatures may prolong the lifetime of Compound I, allowing characterization by UV-visible and possibly RR and cryoradiolysis methods.