CYTOCHROME P450 ACTIVE OXYGEN STRUCTURE AND MECHANISMS

细胞色素 P450 活性氧结构和机制

• 批准号: 6181069
• 负责人: JOHN H DAWSON
• 金额: $ 16.87万
• 依托单位: UNIVERSITY OF SOUTH CAROLINA AT COLUMBIA
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-09-30 至 2002-09-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6181069
• 关键词: chemical bond; chemical reaction; cytochrome P450; electron transport; enzyme structure; enzyme substrate; enzyme substrate analog; hemoprotein structure; nitric oxide synthase; protein structure function

DESCRIPTION: Understanding dioxygen activation by the cytochrome P450 mono-oxygenases requires knowledge of structure at the molecular level. Serving both beneficial and harmful roles in membrane detoxification and carcinogen activation respectively, the cytochromes P450 have been intensively studied. Pseudomonas putida P450-CAM is the best understood P450 in terms of structure and function. The structures of four P450 reaction states (two ferric, deoxyferrous and oxyferrous) have been established. Following second electron transfer, ferric -peroxide and oxo-ferryl adducts have been proposed as intermediates, the latter as the active oxygen catalyst. To observe and spectroscopically characterize such species for the first time, two approaches will be pursued: the use of "slow substrates" blocked at the normal reaction site to impede oxygen transfer and the use of rapid, photointitiated electron transfer to speed electron delivery. This two prong approach will enhance the likelihood of observing the elusive intermediate. In parallel, X-ray absorption spectroscopy will be used to structurally characterize oxo-ferryl states of chloroperoxidase (CPO) and ferric and ferrous states of nitric oxide synthase (NOS). CPO is the only heme protein clearly established to form thiolate-ligated oxo-ferryl intermediates such as that proposed for P450. Like P450, NOS is a thiolate-ligated heme enzyme. Determination of accurate Fe-S(Cys), Fe-N(porph) and Fe=O bond lengths in the CPO and NOS systems will provide structural information directly applicable to the P450 systems also under study. In addition, will be developed the camphor hydroxylating P450-CAM as a versatile enzyme-based model system for mechanistic studies of important P450 reaction types that are difficult to study due to the insolubility and/or complexity of the natural systems. Dr. Dawson will synthesize appropriately functionalized camphor analogues to use as substrates to customize the model system for each reaction type. The three reactions to be examined are: the C-C bond cleaving deformylation reaction, a reaction of great importance in steroid biosynthesis; the second step in the synthesis of the essential biomolecule nitric oxide (NO) in which NO is produced from arginine by nitric oxide synthase; and the P450-catalyzed conversion of nitosamines to NO dealkylated amines, formaldehyde and carinogenic diazoalkanes. The interplay of structure and mechanism is a common theme in all of the proposed work.

描述：了解细胞色素P450对氧气的激活 单加氧酶需要在分子水平上了解结构。 在膜解毒中既有有益的也有有害的作用 致癌物分别被激活后，细胞色素P450被 深入研究。恶臭假单胞菌P450-CAM是人们最了解的 P450的结构和功能。四种P450的结构 反应状态(两种铁、脱氧亚铁和含氧亚铁) 已经成立了。在第二次电子转移后，过氧化铁和 氧铁加合物已被提出作为中间体，后者作为中间体。 活性氧催化剂。观察并从光谱上描述这种 对于首次出现的物种，将采取两种方法：使用 “慢速底物”被阻挡在正常反应部位以阻碍氧气 转移和使用快速的、光引发的电子转移来加速 电子投递。这种双管齐下的方法将增加 观察难以捉摸的中间体。同时，X射线吸收 光谱学将被用来表征氧-铁基的结构 氯过氧化物酶(CPO)与一氧化氮的铁态和亚铁态 合酶(NOS)。Cpo是唯一一种明确形成的血红素蛋白 硫酸盐连接的氧基铁中间体，如为P450建议的中间体。 和P450一样，一氧化氮合酶也是一种硫酸盐连接的血红素酶。准确度的测定 在CPO和NOS体系中，Fe-S(Cys)、Fe-N(Porph)和Fe=O键长将 还提供直接适用于P450系统的结构信息 正在研究中。此外，还将开发樟脑羟化 P450-CAM作为一种通用的基于酶的机制研究的模型系统 重要的P450反应类型难以研究，因为 自然系统的不解性和/或复杂性。道森医生会 合成适当官能化的樟脑类似物用作 底物，为每种反应类型定制模型系统。三位一体 要考察的反应有：C-C键断裂脱甲酰化反应， 在类固醇生物合成中非常重要的反应；第二步 必需生物大分子一氧化氮(NO)的合成 一氧化氮合酶催化精氨酸合成；以及P450催化的 亚硝胺转化为非脱烷基胺、甲醛和 致癌性重氮烷。结构和机制的相互作用是一种 所有拟议工作的共同主题。

项目成果

Sensitizer-linked substrates and ligands: ruthenium probes of cytochrome P450 structure and mechanism.

敏化剂连接的底物和配体：细胞色素 P450 结构和机制的钌探针。

• DOI: 10.1016/s0076-6879(02)57672-2
• 发表时间: 2002
• 期刊: Methods in enzymology
• 作者: Dmochowski,IvanJ;Dunn,AlexanderR;Wilker,JonathanJ;Crane,BrianR;Green,MichaelT;Dawson,JohnH;Sligar,StephenG;Winkler,JayR;Gray,HarryB
• 通讯作者: Gray,HarryB