X-RAY SPECTROSCOPIC STUDIES ON HEME-COPPER OXIDASES

血红素铜氧化酶的 X 射线光谱研究

• 批准号: 2910279
• 负责人: Ninian J Blackburn
• 金额: $ 15.45万
• 依托单位: OREGON GRADUATE INSTITUTE SCIENCE & TECH
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-05-01 至 2001-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2910279
• 关键词: X ray spectrometry; azides; azurin; chemical binding; cyanides; halogens; imidazole; molecular site; nitric oxide; nitrites; oxidoreductase; protein structure function; site directed mutagenesis

DESCRIPTION: Heme-copper oxidases function to couple the energy of oxygen reduction to ATP synthesis in the process of respiration. Thus, these redox enzymes play pivotal roles in aerobic metabolism. The redox processes occur at the metal centers which constitute the active sites of the enzymes and are easily inhibited by small molecules such as cyanides, carbon monoxide and other toxic substances, often with fatal consequences. Understanding the coordination chemistry of the metal centers is a critical step in elucidating the detailed mechanism of respiration and energy transduction within the cell. The recent publication of crystal structures that define the coordination of the metal centers has settled a number of old controversies but has also raised new and even more intriguing questions that can only be addressed by well-focused spectroscopic experiments. This proposal seeks funding to apply X-ray absorption spectroscopy (XAS) to a number of important structural and mechanistic questions highlighted by the crystal structural data. The proposed studies utilize the unique sensitivity of XAS to refine further the metal ion coordination in the oxidized enzyme, and to probe for the first time the coordination present in the reduced enzyme and the catalytic intermediates P and F. The results of these experiments will be used to test the validity of the "histidine cycle,' a recently proposed mechanism for proton pumping. The proposed studies will also investigate in detail the structure of the CuA center in the soluble subunit II fragment, as well as in a number of engineered constructs prepared by "loop-directed" mutagenesis. These experiments follow on from the initial determination from EXAFS of a unique 2.34 A Cu-Cu interaction in CuA. Other experiments are planned that will explore the metal-site coordination in two novel heme-copper oxidases, nitric oxide reductase and azurin oxidase.

描述：血红素铜氧化酶的功能是耦合氧的能量 在呼吸过程中减少ATP的合成。 这些氧化还原 酶在有氧代谢中起关键作用。 氧化还原过程发生 在构成酶的活性位点的金属中心处， 很容易被小分子如氰化物一氧化碳 和其他有毒物质，往往会造成致命的后果。 理解 金属中心的配位化学是 阐明呼吸和能量转换的详细机制 在细胞内。 最近发表的晶体结构， 金属中心的配位解决了许多旧的 争议，但也提出了新的，甚至更有趣的问题 这只能通过聚焦良好的光谱实验来解决。 这 一项提案寻求资金，将X射线吸收光谱（XAS）应用于 一些重要的结构性和机械性问题， 晶体结构数据 拟议的研究利用独特的 XAS的灵敏度进一步细化金属离子配位在 氧化酶，并探测第一次存在的协调， 还原的酶和催化中间体P和F。的结果 这些实验将用于测试“组氨酸”的有效性。 循环，最近提出的质子泵送机制。 拟议 研究还将详细调查CuA中心的结构， 可溶性亚基II片段，以及许多工程化的 通过“环定向”诱变制备的构建体。 这些实验 从EXAFS的独特的2.34 A Cu-Cu的初始测定的后续 在CuA中相互作用。 计划进行的其他实验将探索 两种新的血红素-铜氧化酶-一氧化氮中的金属位配位 还原酶和天青氧化酶。