WHY DO SUBSTRATES REQUIRE CYTOCHROME B5 FOR OXIDATION BY CYTOCHROME P450

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The overall long-term goal of our laboratory is to understand the molecular basis of the effect of cytochrome b5 (cyt b5) on the metabolism of certain substrates by cytochrome P-450 (cyt P-450) and to determine the physiological significance of this reaction. In particular, we are interested in elucidating the molecular basis of the marked stimulatory effect of cyt b5 on the metabolism of the volatile anesthetic, methoxyflurane, by cyt P-450 LM2 (2B4). These studies may eventually lead to the delineation of the etiology and pathophysiology of the postoperative hepatotoxicity attributed to the volatile anesthetics and to the development of safer anesthetics. These studies should also provide important information about the mechanism by which cyt P450 oxidizes its numerous endogenous and xenobiotic substrates. This knowledge should prove valuable in facilitating the design of clinically useful inhibitors of cytochrome P450 for therapeutic applications in fungal diseases, hyperaldosteronism, prostate cancer, benign prostatic hypertrophy and breast cancer. The more immediate short-term goal of this proposal is to use the 23,000 dalton cyt b5-cytochrome c (cyt c) complex as a model for the larger (72,000 dalton) hydrophobic cyt b5-cyt P450 complex and to determine the structure of the model complex using high resolution NMR. The cytochrome b5-cytochrome c complex is a relevant model for the larger hydrophobic complex because cytochrome b5 uses approximately the same site to interact with both cytochrome c and cytochrome P450. Knowledge of the structure of the cyt b5-cyt c complex will provide insights, into 1) the mechanism, 2) pathway of electron transfer, between cyt b5 and its redox partners, and 3) macromolecular recognition and protein dynamics. The appropriate three- and four-dimensional NMR experiments will be conducted on a 500 and 600 MHz NMR spectrometer with the available uniformly 13C/15N labeled bovine cyt b5 in the presence and absence of cyt c. Completion of the proposed NMR experiments should provide us with important structural details about how certain amino acids of cyt b5 facilitate electron transfer with its redox partners, cyt P-450, cyt b5 reductase and cyt c. The computing, graphics and programming facilities at the UCSF Computer Laboratory will be used to construct three-dimensional molecular models based on results from our NMR studies, to display and examine these models, and to calculate the dynamics and interactions of the components in the models; these results should lead to a greater understanding of the structural basis for the requirement for cyt b5 for the metabolism of some substrates by cyt P-450.