PARTICIPATION OF CYTOCHROME B5 IN ANESTHETIC METABOLISM

细胞色素P450(CytsP450)是一个普遍存在的混合功能氧化酶超家族，也被称为大自然的喷灯。在哺乳动物中，细胞色素P45O可能占肝微粒体蛋白的10%，并已知能代谢人类遇到的大多数外来物质，包括药物和致癌物。在纯化的重组系统中，另一种被称为细胞色素b5(Cytb5)的微粒体蛋白可以通过CytP450增加、减少或不影响底物代谢。细胞色素b5如何以及为什么会对细胞色素p45O催化的氧化有如此不可预测的影响，这几十年来一直困扰着研究人员。随着最近内源性化合物和药物的数量显著增加，这些化合物和药物已被证明在cyt b5的存在下提高了它们的新陈代谢，这个问题变得更加有趣和具有生物学意义。本研究的长期目标是了解Cytb5显著刺激细胞色素P450催化的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)催化的模型化合物甲氧基氟烷(MF)、挥发性麻醉剂和苯丙胺的羟基化效率的机制来解决。这些研究可能最终为深入了解由氟烷等挥发性麻醉剂引起的术后肝毒性的病因和病理生理学，以及设计更安全的麻醉药提供帮助。这项提议的短期目标有三个方面。第一个特定目的是研究细胞色素P450还原酶是否能更快地还原含氧亚铁的细胞色素P450 2B4。细胞色素b5对含氧细胞色素P450的还原速度快于细胞色素P45O还原酶，这可以解释细胞色素b5介导的细胞色素P45O周转过程中超氧化物生成减少和产物形成增加的原因。为了进一步探讨Cytb5提高细胞色素P45O的催化效率的机制，我们最近通过鉴定突变的细胞色素P45O和细胞色素b5来描述细胞色素P450-细胞色素b5的蛋白间结合位点，这两个突变的细胞色素P45O和细胞色素b5缺乏与各自氧化还原伙伴的结合能力。第二个具体目标将是根据这些突变蛋白与其氧化还原伙伴结合并在不同氧化状态下进行电子转移反应的能力来表征这些突变蛋白。为了确定哪些细胞色素b5和细胞色素P45O残基在蛋白质间复合体中接触，还将检测一对突变体的结合能力。第三个特定目标是利用定点突变系统地突变这两种蛋白质膜锚上的氨基酸，从而确定Cytb5和CytP45O2B4的膜结合结构域之间的蛋白间结合位点。还提出了评估这种膜内结合作用的特异性的实验。

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.