The cytochromes P450 are a ubiquitous superfamily of mixed function oxidases responsible for the oxidation of a vast number of compounds including the majority of drugs used by humans. In the course of our studies to determine how cytochrome b5 (cyt b5) stimulates the metabolism of substrates such as the anesthetic, methoxyflurane, by cytochrome P450 2B4 (cyt P450) we have recently measured, for the first time, the rate of reduction of a microsomal oxyferrous cyt P450, 2B4, by cyt P450 reductase. This experiment led to the discovery that catalysis by cyt P450 2B4 in the presence of cyt P450 reductase proceeds via a reduced oxyferrous intermediate. In striking contrast, catalysis by cyt P450 2134 in the presence of cyt b5 occurs rapidly without formation of a detectable intermediate allowing less time for unproductive side reactions to occur. The tools developed to measure the rate of reduction of oxyferrous cyt P450 2B4 will now be utilized to further probe the catalytic mechanism of cyt P450 and understand how cyt b5 enhances drug metabolism. The short term goals of this proposal are to elucidate the molecular origin and consequences of the strikingly different effects cyt b5 and cyt P450 reductase have on catalysis by cyt P450 2B4 while at the same time, gaining insight into the reaction mechanism of microsomal cyts P450.
Aim 1. In order to advance our understanding of how cyt b5 and cyt P450 reductase exert their distinctive effects on cyt P450 2B4 catalysis we will trap and identify the intermediates formed during cyt P450 turnover using rapid freeze quench and cryoreduction EPR experiments in collaboration with Dr. Brian Hoffman. The time dependent formation of product during catalysis will be measured using rapid chemical quench techniques.
Aim 2. Since the reduced oxyferrous intermediate which forms in the presence of wild type cyt P450 2B4 and cyt P450 reductase is short lived, three mutant cyts P450 2B4 (T302A, E301Q, F429H) which are expected to accumulate three different longer-lived reaction cycle intermediates [(Fe+3OOH)- (Fe+3OO)= (Fe+2OO)] will be isolated and studied to aid in the identification and characterization of the transient species.
Aim 3. In an effort to gain a detailed molecular understanding of how cyt b5 expedites efficient catalysis by cyt P450 2B4, the amino acids forming interprotein contacts at the binding interface between cyt P450 2B4 and cyt b5 will be identified using site-directed mutagenesis and double-mutant cycle experiments. ? ? ? ? ? ?
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