Heme A and cytochrome oxidase biosynthesis

描述（由申请人提供）：酵母细胞色素c氧化酶（考克斯）是线粒体呼吸链的末端复合物，由12种不同的多肽组成。构成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受三十多个基因控制，这些基因在组装途径的所有阶段进行干预。这些基因的功能一直是并将继续是该项目的重点。在过去的资助期间，我们完成了1）分别在亚基2中CuA中心成熟和血红素A生物合成中起作用的Scolp和Cox15 p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是考克斯组装的结构能力所必需的，4）鉴定了Cox14 p，Mss 51 p，和Coxlp参与调节Coxlp翻译，5）与Pierre Rustin博士合作研究考克斯功能障碍患者。在本申请中，我们建议继续研究考克斯缺陷突变体以更好地理解组装考克斯的过程。这些研究旨在阐明Shylp如何在协调亚基1合成及其用于考克斯组装的调节回路中发挥作用。另一个目的是探讨线粒体膜间金属蛋白Cox23 p、Cox19 p和Pet191 p在考克斯铜中心成熟中的作用。这些研究将检查铜从这些膜间蛋白到Cox17 p的转移，以及它们中的任何一个是否存在于共同的线粒体中？Cox15 p是线粒体的血红素O羟化酶，在我们试图纯化该蛋白质时遇到了一些问题，这阻碍了对Cox15 p的研究。这些困难中最严重的已经得到解决，我们希望获得一个纯化的Cox15 p适合于体外酶的研究，已计划较早。同时，我们将扩展我们的功能分析，以新的和部分特点的COX特异性基因的组合生物化学和遗传学的方法在过去使用。最后，我们将继续与Rustin实验室合作，研究由缺陷的考克斯引起的人类疾病。这些研究将努力使用替代氧化酶来拯救考克斯突变体，并改进目前将人类突变分配给已知考克斯基因的方法。

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.