Role of Subunit III in Cytochrome Oxidase Function

描述（由申请人提供）：细胞色素c氧化酶是关键的能量产生蛋白质之一，其功能是将质子泵送穿过线粒体内膜。该酶的催化核心由三个完整的膜蛋白亚基I、II和III组成。亚基III在电子传递中不起直接作用，但它确实影响亚基I中质子传递途径和质子泵机制的功能。亚基III还防止氧化酶在正常催化周转期间经历快速自杀失活。细胞色素氧化酶的高分辨率结构表明，亚基III与紧密结合的磷脂。细菌Rhodobacter sphaeroides的细胞色素c氧化酶（与线粒体氧化酶密切相关）的诱变和磷脂酶处理表明，这些脂质是将亚基III结合到亚基I所必需的。这是解释了氧化酶的结构，这表明，四个磷脂同时协调的亚基I和亚基III的保守残基。本项目的第一个目的是通过分析旨在中断这些相互作用的定点突变体来研究细胞色素氧化酶的蛋白质-脂质相互作用。这些实验将产生有关如何紧密结合磷脂功能，以促进膜蛋白的结合的信息。这些研究还应该揭示什么类型的亚基间的相互作用的完整的膜蛋白之间的结构信息的传输。第二个目的是研究细胞色素氧化酶中长距离质子转移的过程，使用亚基III和亚基I残基的定点突变。这里的关键问题是质子通路进入位点周围的蛋白质表面如何控制质子转移的速率，以及蛋白质内部的质子传导通路对将该通路连接到另一个亚基的残基的修饰有多敏感。第三个目标是确定自杀失活发生了什么样的结构变化，以了解失活过程的机制。第四个目的是表征铜分子伴侣，这似乎是参与插入到血红素α 3-CuB的细胞色素氧化酶的活性位点的CuB。该组装过程的机制可能不同于蛋白质中其他铜中心的组装，因为细胞色素氧化酶的活性位点埋在亚基I和蛋白质的跨膜区域内。

DESCRIPTION (provided by applicant): Cytochrome c oxidase is one of the key energy-generating proteins that function to pump protons across the inner membrane of mitochondria. The catalytic core of the enzyme is composed of three integral membrane proteins, subunits I, II and III. Subunit III does not play a direct role in electron transfer, but it does affect the function of the proton transfer pathways and the proton pumping mechanism in subunit I. Subunit III also prevents the oxidase from undergoing rapid suicide inactivation during normal catalytic turnover. High resolution structures of cytochrome oxidase show that subunit III is associated with tightly bound phospholipids. Mutagenesis and phospholipase treatment of cytochrome c oxidase of the bacterium Rhodobacter sphaeroides, which is closely related to the mitochondrial oxidase, indicates that these lipids are necessary for binding subunit III to subunit I. This is explained by the oxidase structures which show that four phospholipids are simultaneously coordinated by conserved residues of both subunit I and subunit III. The first aim of this project is to examine the protein-lipid interactions of cytochrome oxidase through the analysis of site-directed mutants designed to interrupt these interactions. These experiments will yield information about how tightly bound phospholipids function to facilitate the binding of membrane proteins. These studies should also reveal what type of inter-subunit interactions between integral membrane proteins are used to transmit structural information. A second aim is to study the process of long-distance proton transfer in cytochrome oxidase, using site-directed mutagenesis of subunit III and subunit I residues. Key questions here are how the protein surface around the entry site of a proton pathway controls the rate of proton transfer and how sensitive is the proton conductive pathway in the interior of the protein to modification of residues that connect the pathway to another subunit. A third aim is to establish what structural changes occur with suicide inactivation in order to understand the mechanism of the inactivation process. A fourth aim is to characterize a copper chaperone that appears to be involved in the insertion of CuB into heme a3-CuB active site of cytochrome oxidase. The mechanism of this assembly process is likely to differ from the assembly of other copper centers in proteins since the active site of cytochrome oxidase is buried within subunit I and within the transmembrane region of the protein.