CYTOCHROME C OXIDASE FROM R SPHAEROIDES

描述 该项目旨在破译分子内转移的机制， 细胞色素c氧化酶（考克斯）中的质子。 作为一个终端成员， 考克斯是线粒体和细菌呼吸链的关键酶， 大多数真核生物的有氧呼吸和能量转换， 细菌 该酶将分子氧还原为水，并利用 这种强放能反应的自由能驱动生电反应， 质子跨膜转移。 的机理 在考克斯中电子和质子的分子内转移和偶联 它们之间的关系一直是生物能量学的中心研究问题 超过50年。 最近，酶的高分辨率结构 已经解决了X射线衍射，它允许集中调查 酶质子泵的分子机制。 有两 连接氧还原血红素-铜的明显通道样结构域 双核中心带负电荷的水相，这些 通道被认为参与氧化还原相关的摄取， 质子的传导 有几个高度保守的质子化 这些通道内的氨基酸残基，最值得注意的是K362、E286和D132 在亚单位I中，已经证明对于 分子内质子转移 将这些残留物替换为 定点诱变强烈降低酶稳态催化活性 活动 在这个项目中，研究人员希望解决部分步骤， 考克斯的分子内质子转移。sphaeroides与援助的 开发的膜电位产生的时间分辨测量 然后以莫斯科的位点特异性突变形式获得酶 从厄巴纳实验室，并确定哪些个别步骤的质子 泵送受到特定质子通道突变的影响 残基 他们希望这些实验能够阐明 考克斯反应机理中的两个质子通道和分子间的相互作用 质子在通道内的迁移机制。

DESCRIPTION The project aims at deciphering the mechanism of intramolecular transfer of protons in cytochrome c oxidase (COX). Being a terminal member of the mitochondrial and bacterial respiratory chain, COX is a key enzyme of aerobic respiration and energy transduction in most eukaryotes and many bacteria. The enzyme reduces molecular oxygen to water and utilizes the free energy of this strongly exergonic reaction to drive the electrogenic translocation of protons across the membrane. The mechanism of intramolecular transfer of electrons and protons in COX and of the coupling between them has been a central research problem in bioenergetics for more than 50 years. Very recently, the high resolution structure of the enzyme has been solved by X-ray diffraction, which allows for focused investigation into the molecular mechanism of proton pumping by the enzyme. There are two apparent channel-like domains connecting the oxygen- reducing heme-copper binuclear center with the negatively charged aqueous phase, and these channels have been suggested to be involved in redox-linked uptake and conduction of protons. There are several highly conserved protonatable amino acid residues within these channels, most notably K362, E286 and D132 in subunit I, that have been shown to be absolutely necessary for intramolecular proton transfer. Replacements of these residues by site-directed mutagenesis strongly reduces enzyme steady state catalytic activity. In this project, the investigators wish to resolve partial steps of intramolecular proton transfer in COX from R. sphaeroides with the aid of time-resolved measurements of membrane potential generation developed in Moscow and then to take site- specific mutant forms of the enzyme available from the Urbana laboratory and determine which individual steps of proton pumping are affected by the mutations in the specific proton channel residues. They hope these experiments will elucidate the specific roles of the two proton channels in the reaction mechanism of COX and the molecular mechanism of proton translocation within the channels.