Catalytic Mechanisms of Heme-Copper Oxidases

血红素铜氧化酶的催化机制

• 批准号: 6605840
• 负责人: MARIAN FABIAN
• 金额: $ 25.66万
• 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 2006-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6605840
• 关键词: Raman spectrometry; active sites; circular dichroism; circular magnetic dichroism; copper; cytochrome oxidase; electron spin resonance spectroscopy; electron transport; enzyme activity; heme oxygenase; hydrogen transport; hydrogen transporting ATP synthase; mitochondrial membrane; oxidation reduction reaction; peroxidases; protonation; stoichiometry; stop flow technique; vesicle /vacuole

DESCRIPTION (provided by applicant): 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 energy 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 one proton translocated per electron transferred. 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, electron 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（由申请人提供）：呼吸氧化酶是膜结合的 催化分子双氧还原的电子转移络合物 水并利用相关的自由能变化来产生跨膜 质子梯度。这种质子梯度是能量的主要来源 人类的生物自由能。近年来，已经很明显的是 大多数呼吸氧化酶都是血红素铜家族的成员 氧化酶超家族。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 亲和配体被结合。细胞色素 c 氧化酶 (CcO) 催化一种 酶促循环 四个当量的铁细胞色素 c 的氧化 位于线粒体内膜的胞质侧，这种氧化是 伴随着四个质子从矩阵空间的消耗 完成两个水分子的形成。消耗的自由能 水的形成不会消散，而是作为跨膜质子保存下来 每个电子转移一个质子易位的化学计量梯度。 氧气完全转化为水是通过特定的氧气进行的 对应于双核离散化学态的中间体 中心。尽管我们已经积累了大量宝贵的知识 近年来，某些含氧中间体的化学性质仍然未得到证实。 关于质子泵浦机制存在争议，而且人们知之甚少 se 以及这些中间体在泵送过程中的参与。这 该提案的目的是确定选定氧的性质 中间体、它们的质子化状态、机理和氧化还原 它们相互转化的化学计量，这些中间体与 质子泵浦活动，以检验守恒的假设 双核中心的电中性是一项基本要求 描述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 和掺入的 CcO 进入囊泡。光谱、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。