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(COX)中的质子。成为该组织的终端成员 线粒体和细菌呼吸链，COX是一种关键酶 大多数真核生物的有氧呼吸和能量转导 细菌。这种酶将分子氧还原为水，并利用 这一强烈的带电反应的自由能驱动生电 质子在膜上的转移。它的作用机制 COX中电子和质子的分子内转移及其耦合 它们之间一直是生物能量学的一个中心研究问题 不只是50年。最近，这种酶的高分辨结构 已经通过X射线衍射得到了解决，这使得可以进行集中的研究 这种酶抽运质子的分子机制。有两个 连接氧还原态血红素-铜的表观通道状结构域 具有带负电荷的水相的双核中心，这些 通道被认为参与了氧化还原相关的摄取和 质子的传导。有几个高度保守的质子化粒子 这些通道中的氨基酸残基，最明显的是K362、E286和D132 在亚基I中，这已被证明是绝对必要的 分子内质子转移。取代这些残留物的是 定点突变大大降低了酶的稳态催化活性 活动。在这个项目中，调查人员希望解决部分步骤 球藻COX分子内质子转移的研究 发展了膜电位产生的时间分辨测量 莫斯科，然后采取位点特定的突变形式的酶可用 从Urbana实验室中获取并确定质子的各个步骤 泵浦受到特定质子通道突变的影响 残留物。他们希望这些实验将阐明这些基因的具体作用 COX反应机理中的两个质子通道及其分子 质子在通道内的转移机制。

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.