CYTOCHROME P450-P450 REDUCTASE INTERACTIONS

细胞色素 P450-P450 还原酶相互作用

• 批准号: 6180250
• 负责人: JULIAN A PETERSON
• 金额: $ 16.84万
• 依托单位: UT SOUTHWESTERN MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 1994
• 资助国家: 美国
• 起止时间: 1994-08-01 至 2002-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6180250
• 关键词: Bacillus megaterium; NAD(P)H dehydrogenase; bacterial proteins; cytochrome P450; electron transport; enzyme mechanism; enzyme model; enzyme structure; ferredoxin; flavin adenine dinucleotide; flavin mononucleotide; flavoproteins; fluorescence spectrometry; iron sulfur protein; isozymes; oxidation reduction reaction; polymerase chain reaction; site directed mutagenesis; thermodynamics

There have been over 700 different genes in the P450 gene-superfamily identified in organisms from bacteria to plants to man. P450s catalyze monooxygenations with the cleavage of molecular oxygen, inserting one oxygen atom into the hydrophobic substrate, while reducing the other to water. This reaction requires two electrons which are transferred from NAD(P)H via a redox partner. P450s can be grouped by their redox- partner requirement: Class I requiring an iron-sulfur protein (ISP) and an NAD(P)H reductase, and Class II requiring an FAD/FMN-containing NADPH reductase. Class I P450s are mitochondrial membrane-associated in eukaryotes and soluble in bacteria, (e.g. P450cam and P450terp), while Class II P450s are endoplasmic reticulum-associated in eukaryotes, with the exception of P450BM-3 from B. megaterium, which is a soluble fusion of a P450 and its reductase. On the liver endoplasmic reticulum, it has been shown that for every 10 to 20 P450 molecules, there is only one reductase molecule. Thus one might ask: What features of a P450 promote reductase binding? What interactions between a P450 and its redox partner allow electron transfer and catalytic activity? and How is this interaction affected by a complex mixture of P450s? Our hypothesis is that interaction and electron transfer between P450s and their redox partners is controlled by a combination of: 1) surface topography, 2) electrostatic charge distribution, and 3) difference in redox potential. To explore the relative contribution of each of these factors, we shall investigate Class I and II P450s, using structurally-known P450cam and P450terp systems as Class I models, and the P450BM-3 system as the Class II model. Moreover, we will investigate the interaction of 3 microsomal Class II P450s both with their eukaryotic redox partner, CPR, and with BMR (the reductase domain of P450BM-3) or its individually-expressed FAD and FMN domains. In preliminary published work we have cloned, expressed, and reconstituted the three individual domains of P450BM-3: the heme, FAD and FMN domains, and will use this as our stepping stone to study the domain/domain interactions in the P450BM-3 system. We will use mutagenesis to determine specific residues involved in this interaction with visible and fluorescence spectroscopy to quantitate binding, stopped-flow kinetics to determine the effect on electron transfer, and product formation. We will extend these studies to include eukaryotic P450s, in combination, and with P450BM-P, to understand competition and regulation of reduction in mixed populations. Finally, we will compare and contrast, P450cam and P450terp using mutagenesis and redox partner swapping.

P450基因超家族中有700多个不同的基因 在从细菌到植物再到人类的有机体中都能识别。P450s催化 插入1的分子氧裂解的单氧反应 氧原子进入疏水基质，同时将另一个还原到 水。这个反应需要两个电子，它们从 NAD(P)H通过氧化还原伙伴。P450可以根据他们的氧化还原进行分组- 合作伙伴要求：第一类需要铁硫蛋白(Isp)和 NAD(P)H还原酶，以及需要含有FAD/FMN的NADPH的II类 还原酶。I类P450是线粒体膜相关蛋白 真核生物，可溶于细菌(如P450cam和P450terp)，而 第二类P450在真核生物中是与内质网相关的，具有 巨型芽孢杆菌的P450BM-3例外，为可溶性融合 一个P450及其还原酶。在肝脏内质网上，它有 已经证明，每10到20个P450分子中，只有一个 还原酶分子。因此，人们可能会问：P450的哪些功能促进了 还原酶结合？P450和它的氧化还原之间有什么相互作用 合作伙伴允许电子转移和催化活性吗？这是怎么回事？ P450的复杂混合物会影响相互作用吗？我们的假设是 P450与其氧化还原反应的相互作用和电子转移 合作伙伴受以下因素控制：1)表面地形，2) 静电电荷分布；3)氧化还原电位的差异。 为了探索这些因素各自的相对贡献，我们将 研究I类和II类P450，使用结构已知的P450和 P450terp系统为I级型号，P450BM-3系统为级 II型。此外，我们还将研究3种微生物体之间的相互作用 第二类P450与它们的真核氧化还原伙伴CPR和 BMR(P450BM-3的还原酶域)或其单独表达的FAD 和FMN结构域。在初步发表的工作中，我们克隆了， 表达并重组了P450BM-3的三个独立结构域： 血红素、FAD和FMN域，并将以此作为我们的垫脚石 研究P450BM-3系统中的结构域/结构域相互作用。我们会 使用突变技术来确定与此相关的特定残留物 与可见光和荧光光谱相互作用进行定量 结合，停流动力学来确定对电子的影响 转移，和产品形成。我们会把这些研究扩展至 包括真核生物P450，并与P450BM-P结合，以 了解混合种群中的竞争和减少的调节。 最后，我们将比较和对比，P450cam和P450terp使用 诱变和氧化还原伙伴互换。