Catalytic Properties of Cytochrome Oxidase

细胞色素氧化酶的催化特性

• 批准号: 6960477
• 负责人: DENIS L. ROUSSEAU
• 金额: $ 30.04万
• 依托单位: ALBERT EINSTEIN COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-09-03 至 2009-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6960477
• 关键词: Raman spectrometry; atomic absorption spectrometry; catalyst; cytochrome oxidase; electron spin resonance spectroscopy; electron transport; enzyme activity; enzyme mechanism; gene expression; hydrogen transport; mitochondrial membrane; mutant; myoglobin; oxidation reduction reaction; oxidative phosphorylation; protein structure function; site directed mutagenesis

DESCRIPTION (provided by applicant): The major goal of this project is to determine the basic functional mechanism of cytochrome c oxidase (CcO), the terminal enzyme in the electron transfer chain. CcO is membrane-bound and has the dual function of reducing O2 to H2O to maintain electron flow to enable oxidative phosphorylation, and of coupling the oxygen reduction chemistry to proton translocation across the inner mitochondrial membrane, to generate a proton gradient. The enzyme plays an indispensable role in mammalian physiology because essentially all vital organs depend on aerobic metabolism. Despite years of intensive studies on CcO, the understanding of its catalytic processes is still incomplete and its mechanism of proton translocation remains unclear. We propose a variety of experiments to delineate the oxygen reduction chemistry and aim to shed new light on the coupled proton translocation. In order to identify the catalytic intermediates, new rapid mixing, laser photolysis and freeze trapping techniques will be applied to studies of both the mammalian and the bacterial forms of the enzyme. Full characterization will be done with resonance Raman scattering, optical absorption and electron paramagnetic resonance spectroscopies. One of the major focuses will be to determine the properties of the still controversial short-lived peroxo and ferryl intermediates. Resonance Raman and optical absorption spectroscopy will also be used in conjunction with a home-built continuous flow apparatus to monitor the time-dependent population of the reactive intermediates present during single as well as multiple turnover of the enzyme. To determine the role of specific residues in the functional processes of the enzyme, site directed mutants of CcO from bacteria will be expressed and analyzed by this multifaceted approach. In addition, the oxygen chemistry in a new class of model complexes, a mutant myoglobin with a binuclear center engineered in it to mimic the catalytic site of CcO, will be studied to elucidate the role of the binuclear heme-copper center. The energy transduction and proton translocation mechanism of CcO will be constructed, tested and refined.

描述（由申请人提供）：本项目的主要目标是确定细胞色素c氧化酶（CcO）的基本功能机制，CcO是电子传递链中的末端酶。CcO是膜结合的，并且具有将O2还原成H2O以维持电子流从而使氧化磷酸化成为可能，以及将氧还原化学偶联到穿过线粒体内膜的质子易位以产生质子梯度的双重功能。这种酶在哺乳动物生理学中起着不可或缺的作用，因为基本上所有重要器官都依赖于有氧代谢。尽管多年来对CcO进行了深入的研究，但对其催化过程的了解仍然不完全，其质子转运机制仍然不清楚。我们提出了各种各样的实验来描绘氧还原化学，旨在揭示耦合质子易位的新的光。为了确定催化中间体，新的快速混合，激光光解和冷冻捕获技术将应用于研究哺乳动物和细菌形式的酶。将用共振拉曼散射、光吸收和电子顺磁共振光谱进行全面表征。其中一个主要的重点将是确定仍然有争议的短寿命过氧和铁基中间体的性能。共振拉曼和光学吸收光谱也将与自制的连续流动装置一起使用，以监测酶的单次和多次周转期间存在的反应性中间体的时间依赖性群体。为了确定特定残基在酶的功能过程中的作用，将通过这种多方面的方法表达和分析来自细菌的CcO的定点突变体。此外，在一类新的模型配合物，一个突变的肌红蛋白与双核中心工程，以模仿CCO的催化位点的氧化学，将研究阐明双核血红素铜中心的作用。CcO的能量传递和质子转运机制将被构建、测试和完善。