CYTOCHROME P450-P450 REDUCTASE INTERACTIONS

细胞色素 P450-P450 还原酶相互作用

• 批准号: 2706351
• 负责人: JULIAN A PETERSON
• 金额: $ 18.3万
• 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/2706351
• 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 从细菌到植物再到人类的生物体中都有P450的存在。 单氧化与分子氧的裂解，插入一个 氧原子进入疏水性基质，同时将另一个还原为 水 这个反应需要两个电子， NAD（P）H通过氧化还原配偶体。 P450可以按其氧化还原进行分组- 伴侣要求：I类需要铁硫蛋白（ISP）， NAD（P）H还原酶，II类需要含FAD/FMN的NADPH 还原酶。 I类P450是线粒体膜相关的， 真核生物和可溶于细菌，（例如P450 cam和P450 terp），而 II类P450在真核生物中与内质网相关， B的P450 BM-3除外。megaterium是一种可溶性融合物 P450和它的还原酶。 在肝内质网上， 每10到20个P450分子， 还原酶分子 因此，有人可能会问：P450促销的特点是什么？ 还原酶结合？ P450和它的氧化还原反应 伴侣允许电子转移和催化活性？这是怎么回事 P450的复杂混合物影响的相互作用？ 我们的假设是 P450与其氧化还原反应之间相互作用和电子转移 合作伙伴是由以下组合控制：1）表面形貌，2） 静电荷分布; 3）氧化还原电位差。 为了探索这些因素中每一个的相对贡献，我们将 研究I类和II类P450，使用结构已知的P450凸轮， P450 terp系统作为I类型号，P450 BM-3系统作为 II模型。 此外，我们还将研究3种微粒体之间的相互作用。 II类P450与它们的真核氧化还原配偶体CPR，以及与 BMR（P450 BM-3的还原酶结构域）或其单独表达的FAD 和FMN域。在我们克隆的初步发表的工作中， 表达并重建P450 BM-3的三个单独的结构域： 血红素，FAD和FMN域，并将使用此作为我们的垫脚石 研究P450 BM-3系统中结构域/结构域的相互作用。 我们将 使用诱变来确定参与这一过程的特定残基 与可见光和荧光光谱相互作用， 结合，停流动力学，以确定对电子 转移和产物形成。 我们将把这些研究扩展到 包括真核P450，与P450 BM-P组合， 理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，P450 cam和P450 terp使用 诱变和氧化还原配偶体交换。