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 催化分子双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 梯度 这种质子梯度是生物能量的主要来源 人类的自由能。 近年来，很明显，大多数 呼吸氧化酶是血红素铜氧化酶家族的成员 超级家庭。 该家族的成员拥有独特的双金属中心 由血红素加铜组成；在这个中心，分子氧减少并且很高 亲和配体结合细胞色素 c 氧化酶 (CcO) 催化一种 酶促循环 四当量铁细胞色素 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 空间完成两个水分子的形成。 自由能 形成水时所消耗的能量并没有消散，而是作为水的形式保存下来 化学计量为一个质子的跨膜质子梯度 每个电子转移易位；第二个过程称为 该酶的质子泵活性。 氧气完全转化 水通过特定的氧中间体进行，对应于 双核中心的离散化学状态。 尽管身体庞大 近年来积累的宝贵化学知识 这些含氧中间体中某些的性质仍然存在争议并且非常有争议。 人们对质子泵浦机制本身知之甚少 这些中间体参与泵送过程。 的目标 该提案旨在确定选定的含氧中间体的性质， 它们的质子化状态、机理和氧化还原化学计量 相互转化，这些中间体与质子泵浦的关系 活性，检验电中性守恒定律的假设 双核中心是一个基本要求，并表征 配体相互作用的机制。 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 并将 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 用于实现这些目标。