The cytochromes P450 (cyts P450) are an ubiquitous superfamilv of mixed function oxidases which are also known as Mother Nature's blowtorch. In mammals, cyts P45O may account for up to 10% of the hepatic microsomal proteins and are known to metabolize a. majority of the xenobiotics including drugs and carcinogens encountered by man. In purified reconstituted systems, a second microsomal protein known as cytochrome b5 (cyt b5) 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 P45O 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 cyt b5 increases the efficiency of the cyt P450 2B4 (LM2) catalyzed hydroxylation of the model compounds methoxyflurane (MF), a volatile anesthetic, and benzphetamine. These studies may eventually provide insight into the etiology and pathophysiology of the postoperative hepatotoxicity attributed to the volatile anesthetics, such as halothane, and the design of safer anesthetics. The short-term goals of this proposal are three-fold. The first specific aim is to investigate whether cyt b5 or cyt P450 reductase reduces oxyferrous cyt P450 2B4 more rapidly. More rapid reduction of oxyferrous cyt P450 by cyt b5 than by cyt P45O reductase would explain the cyt b5-mediated decrease in the production of superoxide and increase in product formation during cyt P45O turnover. To further probe the mechanism by which cyt b5 increases the catalytic efficiency of cyt P45O, we have recently delineated the cyt P450-cyt b5 interprotein binding site by identifying mutant cyts P45O and cyts b5 which are deficient in their ability to bind to their respective redox partners. The second specific aim will be to characterize these mutant proteins with respect to their ability to bind their redox partners and undergo electron transfer reactions in different oxidation states. In an effort to determine which cyt b5 and cyt P45O residue are in contact in the interprotein complex, the ability of pairs of mutants to bind will also be examined. The third specific aim will be to delineate the interprotein binding site between the membrane binding domains of cyt b5 and cyt P45O 2B4 using site-directed mutagenesis to systematically mutate amino acids in the membrane anchors of these two proteins. Experiments are proposed which will also evaluate the specificity of this intramembrane binding interaction.
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