CYTOCHROME P450 ACTIVE OXYGEN STRUCTURE AND MECHANISMS

细胞色素 P450 活性氧结构和机制

• 批准号: 2023396
• 负责人: JOHN H DAWSON
• 金额: $ 15.9万
• 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 2001-09-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2023396
• 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 激活双氧 单加氧酶需要分子水平的结构知识。 在膜解毒和解毒过程中发挥有益和有害的作用 分别激活致癌物，细胞色素P450已被 深入研究。 恶臭假单胞菌 P450-CAM 最好理解 P450在结构和功能方面。 四个P450的结构 反应状态（两种铁、脱氧亚铁和含氧亚铁） 已确立的。 第二次电子转移后，过氧化铁和 氧代铁基加合物已被提议作为中间体，后者作为 活性氧催化剂。 观察并用光谱表征此类 首次物种，将采用两种方法：使用 “慢底物”被阻挡在正常反应位点以阻碍氧气 转移和使用快速光引发电子转移来加速 电子传递。 这种两管齐下的方法将提高 观察难以捉摸的中间体。 同时，X 射线吸收 光谱将用于结构表征的氧代铁基状态 氯过氧化物酶 (CPO) 和一氧化氮的铁态和亚铁态 合酶（NOS）。 CPO 是唯一明确形成的血红素蛋白 硫醇盐连接的氧代铁基中间体，例如针对 P450 提出的中间体。 与 P450 一样，NOS 是一种硫醇盐连接的血红素酶。 测定准确 CPO 和 NOS 系统中的 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，还将开发樟脑羟基化 P450-CAM 作为一种多功能酶基模型系统，用于机械研究 重要的 P450 反应类型由于以下原因而难以研究 自然系统的不溶性和/或复杂性。 道森博士将 合成适当功能化的樟脑类似物用作 底物来为每种反应类型定制模型系统。 三个 要检查的反应是：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 重要生物分子一氧化氮 (NO) 的合成，其中 NO 是 由一氧化氮合酶从精氨酸产生；和P450催化的 将亚硝胺转化为NO脱烷基胺、甲醛和 致癌重氮烷。 结构和机制的相互作用是 所有拟议工作的共同主题。