Role of Subunit III in Cytochrome Oxidase Function

描述(申请人提供)：细胞色素c氧化酶是关键的能量产生蛋白之一，其功能是将质子泵过线粒体的内膜。该酶的催化核心由三个完整的膜蛋白组成，即亚基I、亚基II和亚基III。亚基III在电子转移中不起直接作用，但它确实影响质子转移途径的功能和亚基I中的质子泵机制。亚基III还可以防止酶在正常的催化周转过程中发生快速自杀性失活。细胞色素氧化酶的高分辨结构表明，亚基III与紧密结合的磷脂有关。球形红杆菌细胞色素c氧化酶的诱变和磷脂酶处理表明，这些脂类是结合亚基III和亚基I所必需的，这是由氧化酶结构解释的，该结构表明四种磷脂同时被亚基I和亚基III的保守残基所协调。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。这些实验将提供关于结合磷脂的功能如何紧密地促进膜蛋白结合的信息。这些研究还应该揭示完整的膜蛋白之间的哪种亚基间相互作用被用来传递结构信息。第二个目的是利用亚基III和亚基I残基的定点突变，研究细胞色素氧化酶中质子的长距离转移过程。这里的关键问题是质子途径进入部位周围的蛋白质表面如何控制质子转移的速度，以及蛋白质内部的质子传导途径对连接该途径与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会发生什么结构变化，以了解失活过程的机制。第四个目的是鉴定一种铜伴侣，它似乎与幼崽插入细胞色素氧化酶活性部位的血红素A3-幼崽有关。这种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部位埋在蛋白质的亚基I和跨膜区内。

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.