Date of Award

Spring 2015

Document Type


Degree Name

Doctor of Philosophy (PhD)



First Advisor

Fiedler, Adam

Second Advisor

Ryan, Michael D.

Third Advisor

Sem, Daniel S.


The Cytochromes P450 are monooxygenases which use dioxygen and endogenous reducing equivalents to generate highly reactive intermediates capable of performing several types of oxidative transformations, most commonly, hydroxylations via the so-called Compound I species. There are two main kinds of mammalian P450s: those functioning in drug metabolism and those that facilitate steroid biosynthesis. Steroidogenic P450s participating in the biosynthesis of steroid hormones are involved in a number of different pathways. CYP11A1, one of the steroidogenic P450s also known as P450scc, is the enzyme that converts cholesterol to pregnenolone through side chain cleavage between carbon 20 and 22. The overall process is a three step reaction. The two intermediates, 22(R)-hydroxycholesterol and 20R,22R-dihydroxycholesterol, bind more tightly than either cholesterol or pregnenolone. One of the most interesting aspects of the enzymatic cycle is that, while the first two reactions involve substrate hydroxylations, presumably proceeding through the Compound I species, the third reaction involves a side chain cleavage reaction that has been proposed to occur through Compound I attack on the diol fragment of the 20R,22R-dihydroxycholesterol.The present work employs resonance Raman (rR) spectroscopy to structurally characterize the enzyme active site structure. Also, in order to obtain a generally useful calibration procedure to permit reliable rR-based evaluation of the percentage of HS and LS population for CYP11A1 and other P450s, a method was developed to relate HS/LS populations to measured intensities of spin state markers based on well-behaved CYP101 reference samples. These cross section values from P450cam high spin and low spin are used to determine the spin population in CYP11A1 complex with three different substrates and evaluate the effect of Adx binding. The work also clarifies certain confusing issues that had arisen from previously published spectroscopic studies of ferric and ferrous-CO states. Most significantly, the present work provides the first rR spectroscopic characterization of the oxy adducts for the three substrate-bound forms, providing some insight into the active intermediates involved in the three sequential reactions.