PARTICIPATION OF CYTOCHROME B5 IN ANESTHETIC METABOLISM

细胞色素P450 （cyts P450）是一种普遍存在的混合功能氧化酶超家族，也被称为大自然母亲的喷灯。在哺乳动物中，P45O细胞可能占肝微粒体蛋白的10%，并且已知代谢大多数人类遇到的外源物，包括药物和致癌物。在纯化的重组系统中，称为细胞色素b5 （cyt b5）的第二微粒体蛋白可以增加，减少或对cyt P450的底物代谢没有影响。几十年来，cyt b5如何以及为什么会对cyt P45O催化氧化产生如此不可预测的影响一直困扰着研究人员。随着最近内源性化合物和药物数量的显著增加，这些化合物和药物已被证明在存在cyt b5的情况下可以增加它们的代谢，这个问题变得更加有趣和具有生物学意义。本提案的长期目标是了解cyt b5显著刺激cyt P450催化某些底物代谢的分子基础，并确定该反应的生理意义。这个问题将通过阐明cyt b5提高cyt P450 2B4 （LM2）催化模型化合物甲氧基氟醚（MF）（一种挥发性麻醉剂）和苯非他明的羟基化效率的机制来解决。这些研究可能最终为挥发性麻醉药（如氟烷）术后肝毒性的病因和病理生理学以及更安全麻醉药的设计提供见解。这项提议的短期目标有三个方面。第一个具体目的是研究cyt b5或cyt P450还原酶是否能更快地还原氧化亚铁cyt P450 2B4。cyt b5比cyt P45O还原酶更快地还原氧化亚铁细胞P450，这可以解释在cyt P45O转换过程中，cyt b5介导的超氧化物产生减少和产物形成增加。为了进一步探索cyt b5提高cyt P45O催化效率的机制，我们最近通过鉴定缺乏与其各自氧化还原伙伴结合能力的突变cyt P45O和cyt b5，描绘了cyt P450-cyt b5蛋白间结合位点。第二个具体目标将是表征这些突变蛋白与它们的氧化还原伙伴结合的能力，并在不同的氧化状态下进行电子转移反应。为了确定哪些cyt b5和cyt P45O残基在蛋白间复合体中接触，还将检查对突变体结合的能力。第三个具体目标将是描述cyt b5和cyt P45O 2B4膜结合域之间的蛋白间结合位点，使用位点定向诱变系统地突变这两种蛋白的膜锚点中的氨基酸。实验也将评估这种膜内结合相互作用的特异性。

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.