CATALYTIC MECHANISM OF HEME/COPPER OXIDASES

血红素/铜氧化酶的催化机制

• 批准号: 6386697
• 负责人: Graham A. Palmer
• 金额: $ 16.74万
• 依托单位: RICE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-06-01 至 2002-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6386697
• 关键词: Raman spectrometry; active sites; circular dichroism; circular magnetic dichroism; copper; cytochrome oxidase; electron spin resonance spectroscopy; enzyme mechanism; heme oxygenase; hydrogen transport; hydrogen transporting ATP synthase; laboratory rat; ligands; mitochondrial membrane; peroxidases; stoichiometry; stop flow technique; vesicle /vacuole

DESCRIPTION: Respiratory oxidases are membrane-bound electron-transfer complexes which catalyze the reduction of molecular dioxygen to water and use the associated free energy changes to generate a transmembrane proton gradient This proton gradient is the primary source of energy for biological free energ in humans. In recent years it has become apparent that most respiratory oxidases are members of a single family, the heme-copper oxidase super-family. Members of this family have a unique bimetallic center composed of heme plus copper; at this center dioxygen is reduced and high affinity ligands are bound Cytochrome c oxidase (CcO) catalyzes in one enzymatic cycle the oxidation of four equivalents of ferrocytochrome c located on the cytosolic side of the inner mitochondrial membrane and this oxidation is accompanied by the consumption of four protons from the matrix space to complete the formation of two water molecules. The free energy expended in the formation of water is not dissipated but conserved as a trans-membrane proton gradient with a stoichiometry of one proton translocated per electron transferred; this second process is called the proton pumping activity of this enzyme. The complete conversion of oxygen to water proceeds through specific oxy intermediates corresponding to discrete chemical states of the binuclear center. Despite a large body of valuable knowledge that has been accumulated in recent years the chemical nature of certain of these oxy intermediates is still controversial and very little is known about the proton pumping mechanism per se and the involvement of these intermediates in the pumping process. The objective of this proposal is to establish the nature of selected oxy intermediates, their protonation state, the mechanism and the redox stoichiometry of their interconversion, the relation of these intermediates to the proton pumping activity, to test the hypothesis that the conservation of electroneutrality at the binuclear center is a fundamental requirement and to characterize the mechanism of ligand interaction(s). To address these problems we plan to use isolated mitochondria, purified bovine CcO and CcO incorporated into vesicles. Optical spectroscopy, electon paramagnetic resonance with rapid quenching kinetics, magnetic and natural circular dichroism, resonance Raman spectroscopy, stopped-flow kinetics, and several biochemical methods will be used to accomplish these goals.

描述：呼吸氧化酶是膜结合电子转移酶 催化分子氧还原成水的络合物， 利用相关的自由能变化产生跨膜质子 这种质子梯度是生物能量的主要来源。 人体内的自由能量 近年来，人们发现， 呼吸氧化酶是一个单一家族的成员，血红素铜氧化酶 超级家庭 这个家庭的成员有一个独特的心脏 由血红素加铜组成;在这个中心，分子氧被还原， 细胞色素c氧化酶（CcO）催化一种 酶循环四当量的亚铁细胞色素c的氧化 位于线粒体内膜的胞质侧， 氧化伴随着基质中四个质子的消耗 空间来完成两个水分子的形成。 自由能 在水的形成中消耗的能量不是消散的，而是作为一种能量保存下来的。 一个质子化学计量的跨膜质子梯度 每个电子转移的转移;这第二个过程被称为 这种酶的质子泵活性。 氧气的完全转化 通过特定的含氧中间体， 双核中心的离散化学状态。 尽管有大量的 近年来积累的宝贵知识， 某些这些含氧中间体的性质仍然是有争议的， 关于质子泵送机制本身和质子泵的性质知之甚少。 在泵送过程中这些中间体的参与。 的目标 该提议是为了确定所选氧中间体的性质， 它们的质子化状态，它们的质子化机理和它们的氧化还原化学计量比。 这些中间体与质子泵的关系 活动，以检验假设，即电中性守恒 在双核中心是一个基本的要求，并表征 配体相互作用机制。 为了解决这些问题，我们计划 使用分离的线粒体、纯化的牛CcO和CcO掺入 囊泡 光谱学，电子顺磁共振快速 淬灭动力学，磁性和自然圆二色性，共振拉曼 光谱学、停流动力学和几种生化方法将被 用于实现这些目标。