Heme A and cytochrome oxidase biosynthesis

描述（由申请人提供）：酵母细胞色素c氧化酶（COX）是线粒体呼吸链的末端复合体，由12种不同的多肽组成。构成该复合物催化核心的三个亚基由线粒体基因编码。其余8个亚基是核基因组的产物。这种重要酶的生物发生是由30多个基因控制的，这些基因在组装途径的所有阶段都起作用。这些基因的功能一直是并将继续是这个项目的重点。在过去的资助期间，我们完成了1)分别在亚基2的CuA中心成熟和血红素A的生物合成中起作用的Scolp和COX 15p的研究，2)鉴定并研究了4个新的COX特异性基因的功能，3)表明细胞色素c是COX组装的结构能力所必需的，4)鉴定了Cox14p， Mss51p和Coxlp的三联物，参与调节Coxlp的翻译。5)与Pierre Rustin博士合作进行COX功能障碍患者的研究。在目前的应用中，我们建议继续研究COX缺乏突变体，以更好地了解COX组装的过程。这些研究将旨在阐明Shylp在协调亚基1合成及其用于COX组装的调控回路中如何起作用。另一个目的是探索线粒体膜间金属蛋白Cox23p、Cox19p和pet191p在COX铜中心成熟中的作用。这些研究将检查铜从这些膜间蛋白转移到Cox17p，以及它们是否存在于一个共同的络合物中。线粒体的血红素O羟化酶Cox15p的研究由于我们试图纯化该蛋白时遇到的问题而受到阻碍。这些困难中最严重的已经解决，我们期望获得纯化的Cox15p，适用于先前计划的体外酶学研究。同时，我们将通过结合过去使用的生化和遗传方法，将我们的功能分析扩展到新的和部分表征的cox特异性基因。最后，我们将继续与Rustin实验室合作，研究由COX缺陷引起的人类疾病。这些研究将努力使用一种替代氧化酶来拯救COX突变体，并改进目前将人类突变分配给已知COX基因的方法。

DESCRIPTION (provided by applicant): Yeast cytochrome c oxidase (COX), the terminal complex of the mitochondrial respiratory chain, is composed of 12 different polypeptides. The three subunits constituting the catalytic core of the complex are encoded by mitochondrial genes. The remaining 8 subunits are products of the nuclear genome. Biogenesis of this important enzymes is governed by more than three dozen genes that intercede at all stages of the assembly pathway. The functions of these genes have been and continue to be the focus of this project. During the past grant period we have 1) completed studies on Scolp and Cox15p that function in maturation of the CuA center in subunit 2 and biosynthesis of heme A, respectively, 2) identified and studied the functions of 4 new COX-specific genes, 3) shown that cytochrome c is required in a structural capacity for COX assembly, 4) identified a ternary complex of Cox14p, Mss51p, and Coxlp involved in regulation of Coxlp translation, 5) collaborated with Dr. Pierre Rustin on studies of patients with COX dysfunctions. In the present application we propose to continue investigations of COX deficient mutants to gain a better understanding of the process by which COX is assembled. These studies will aim to clarify how Shylp functions in the regulatory circuit that coordinates the synthesis of subunit 1 to its utilization for COX assembly. Another aim is to explore the roles of the mitochondrial intermembrane metallo-proteins Cox23p, Cox19p, and Pet191 p in maturation of the copper centers of COX. These studies will examine copper transfer from these intermembrane proteins to Cox17p and whether any of them are present in a common complexe(s)? Studies on Cox15p, the heme O hydroxylase of mitochondria, were hampered by problems encountered in our attempts to purify the protein. The most serious of these difficulties have been resolved and we expect to obtain a purified Cox15p suitable for the in vitro enzymatic studies that had been planned earlier. Concurrently, we will extend our functional analyses to new and partially characterized COX-specific genes by combined biochemical and genetic approaches used in the past. Finally, we will continue to collaborate with the Rustin lab on studies of human disorders stemming from defective COX. These studies will strive to use an alternative oxidase to rescue COX mutants and to improve on current methods for assigning human mutations to known COX genes.