Cyt 5 can increase, decrease or have no effect on substrate metabolism by cyt P450. How and why cyt b5 can have such an unpredictable effect on cyt P450 catalyzed oxidations has puzzled researchers for decades. With the recent marked increase in the number of endogenous compounds and drugs that have been shown to increase their metabolism in the presence of cyt b5, this question is becoming more interesting and biologically relevant. The long term objective of this proposal is to understand the molecular basis of the marked stimulation of the cyt P450 catalyzed metabolism of certain substrates by cyt b5 and to determine the physiological significance of this reaction. The problem will be addressed by elucidating the mechanism by which the model compound methoxyflurane (MF), a volatile anesthetic, induces the requirement for cyt b5 for its metabolism by cyt P450 2B4 (LM2). These studies may eventually lead to the delineation of the etiology and pathophysiology of the postoperative hepatotoxicity attributed to the volatile anesthetics, and should contribute to our understanding of the mechanism of oxidation by cyt P450. The short term goals of this proposal are three-fold. The first specific aim, using stopped-flow spectrophotometry, is to investigate the effect of the substrates, MF and benzphetamine on the binding of cyt P450 reductase and cyt b5 to oxyferrous cyt P450 and on the rate of reduction of oxyferrous cyt P450 by these two proteins. The rate at which the product of both substrates is released from the substrate binding site will also be investigated. The interpretation of these studies will be greatly facilitated by calculations of the spectra, electronic structure and spin distribution of stable and transient intermediates in the cyt P450 reaction cycle, which will be performed in collaboration with Dr. Gilda Loew. The second specific aim is to continue our mutation studies into the role of specific amino acids of cyt b5 in the oxidation of MF by cyt P450. Of major interest will be two groups of residues, the acidic residues around the heme of cyt b5 which are involved in binding to its soluble redox partners, and the amino acids on the putative inter-protein binding surface. The third specific aim will be to locate the interprotein binding site on cyt P450 for cyt b5. This will be accomplished using a model of cyt P450 2B4 to select which amino acids on the surface of cyt P450 2B4 might be involved in interactions with cyt b5. The selected amino acids will then be systematically mutated to alanine using the technique of alanine scanning. The ability of the mutant cyt P450 molecules to bind cyt b5 and to function in electron transfer with these proteins will be evaluated.
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