Catalytic Properties of Cytochrome Oxidase

项目描述（申请人提供）：本项目主要目的是确定电子传递链末端酶细胞色素c氧化酶（CcO）的基本功能机制。CcO是膜结合的，具有将O2还原为H2O以维持电子流以实现氧化磷酸化的双重功能，并将氧还原化学与穿过线粒体内膜的质子易位偶联以产生质子梯度。这种酶在哺乳动物生理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。尽管对CcO进行了多年的深入研究，但对其催化过程的了解仍然不完整，其质子易位的机制也不清楚。我们提出了各种实验来描述氧还原化学，旨在揭示耦合质子易位的新线索。为了鉴定催化中间体，新的快速混合、激光光解和冷冻捕获技术将应用于该酶的哺乳动物和细菌形式的研究。完整的表征将完成共振拉曼散射，光学吸收和电子顺磁共振光谱。主要焦点之一将是确定仍有争议的短寿命过氧和铁基中间体的性质。共振拉曼光谱和光学吸收光谱也将与自制的连续流动装置一起使用，以监测在酶的单次和多次周转期间存在的反应中间体的时间依赖性种群。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细菌CcO的位点定向突变体。此外，将研究一类新的模型复合物的氧化学性质，即具有双核中心的突变型肌红蛋白，以模拟CcO的催化位点，以阐明双核血红素-铜中心的作用。构建、测试和完善CcO的能量转导和质子易位机制。

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.