CYTOCHROME P450 ACTIVE OXYGEN STRUCTURE AND MECHANISMS

细胞色素 P450 活性氧结构和机制

• 批准号: 2796770
• 负责人: JOHN H DAWSON
• 金额: $ 16.06万
• 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/2796770
• 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的双氧活化 单加氧酶需要分子水平的结构知识。 在膜解毒中起有益和有害的作用， 致癌物激活分别，细胞色素P450已被 深入研究。 恶臭假单胞菌P450-CAM是最好理解的 P450在结构和功能方面。 四种P450的结构 反应状态（两个铁，脱氧亚铁和氧亚铁）已被 确立了习 在第二次电子转移后，过氧化铁和 已经提出氧代-铁基加合物作为中间体，后者作为 活性氧催化剂 为了观察和光谱表征这种 物种的第一次，两种方法将被追求：使用 “慢底物”在正常反应位点受阻，阻碍氧 转移和使用快速，光引发的电子转移速度 电子传递 这种双管齐下的做法将提高 观察难以捉摸的中间体。 同时，X射线吸收 光谱学将用于结构表征氧代铁基状态的 氯过氧化物酶（CPO）和一氧化氮的铁态和亚铁态 合成酶（NOS）。 CPO是唯一一种明确确定形成 硫醇盐-连接的氧代-铁基中间体，例如为P450提出的。 与P450一样，NOS是一种巯基连接的血红素酶。 准确度测定 在CPO和NOS体系中，Fe-S（Cys）、Fe-N（porph）和Fe=O键长将 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作为一种多功能酶模型系统，用于 重要的P450反应类型难以研究，因为 自然系统的不溶性和/或复杂性。 道森医生会 合成适当官能化樟脑类似物，用作 基质，为每种反应类型定制模型系统。 三 待检测的反应是：C-C键断裂脱乙酰基反应， 在类固醇生物合成中非常重要的反应; 一氧化氮（NO）是一种重要的生物分子， 由精氨酸通过一氧化氮合酶产生;和P450催化的 将亚硝胺转化为NO脱烷基化胺、甲醛和 致癌重氮烷 结构和机制的相互作用是一个 所有建议的工作中的共同主题。