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：通过细胞色素P450了解二氧化物激活 单氧酶需要在分子水平上了解结构。 在膜排毒和有害作用和有害作用 致癌物激活分别是细胞色素P450是 深入研究。 假单胞菌Putida P450-CAM是最好的理解 P450在结构和功能方面。 四个P450的结构 反应状态（两个铁，脱氧和氧气） 已确立的。 在第二个电子转移之后，铁氧化铁和 已经提出了氧加合物作为中间体，后者作为中间体 活性氧催化剂。 观察并在光谱上表征这样的表征 物种首次将采用两种方法：使用 在正常反应部位阻塞的“慢底物”阻碍了氧气 转移和使用快速，光刺激的电子传输到速度 电子传递。 这种两种插入方法将增强 观察难以捉摸的中间体。 同时，X射线吸收 光谱法将用于在结构上表征氧气的效果 一氧化氮的氯氧化酶（CPO）以及铁和亚铁态 合酶（NOS）。 CPO是唯一明确形成的血红素蛋白 硫醇含量的氧素弗雷素中间体，例如针对P450提出的中间体。 像P450一样，NOS是一种硫醇含量的血红素酶。 确定准确 CPO和NOS系统中的Fe-S（Cys），Fe-N（Porph）和Fe = O键长度将 还提供直接适用于P450系统的结构信息 正在研究。 此外，还将开发樟脑羟基化 P450-CAM作为一种基于多功能酶的模型系统，用于机械研究 重要的P450反应类型，由于 自然系统的不溶性和/或复杂性。 道森博士 合成适当功能化的樟脑类似物 为每种反应类型自定义模型系统的基板。 三个 要检查的反应是：C-C键裂解畸形反应， 在类固醇生物合成中非常重要的反应；第二步 基本生物分子一氧化氮（NO）的合成，其中否IS 用一氧化氮合酶从精氨酸产生；和P450催化 硝基胺转化为无脱甲胺，甲醛和 突发性重氮基因。 结构和机制的相互作用是 在所有拟议的工作中的共同主题。