Date of Award
Fall 2012
Document Type
Dissertation
Degree Name
Doctor of Philosophy (PhD)
Department
Chemistry
First Advisor
Kincaid, James R.
Second Advisor
Ryan, Michael D.
Third Advisor
Sem, Daniel S.
Abstract
Cytochromes P450 (P450s) have been the subject of intense research for over six decades. Though it is widely accepted that a highly reactive Fe(IV)=O π-cation radical, or the so called 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. In this work, chemically inert substrates of P450s have been utilized in a new approach to capture and stabilize this transient intermediate and characterize it with resonance Raman (RR) spectroscopy, which is a well established tool for studying heme proteins. Specifically, perfluorodecanoic acid has been utilized as an inert surrogate substrate of a thermophilic cytochrome P450 designated CYP119 and RR and cryoradiolysis methods were employed to characterize the enzymatic intermediates under turnover conditions.
In a separate project, a recent and more efficient approach for the isotopic labeling of the prosthetic group in heme proteins has been exploited to produce a 13C labeled analogue of the soluble bacterial cytochrome P450cam (P450cam). Briefly, the HU227 strain of E. coli that 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. This system has been utilized as proof of principle for the strategy of defining active site structure in mammalian cytochromes P450 using NMR methods to furnish necessary experimental restrictions in docking routines, which are commonly employed in determining the relative affinities of drug candidates. Noting that few crystal structures of substrate bound complexes of drug metabolizing P450s exist, a truncated CYP2D6 gene has been designed following a recently published procedure and efforts were made to heterologously express a selectively13C enriched analogue of this important drug metabolizing enzyme.