Heme A and cytochrome oxidase biosynthesis

描述（申请人提供）：细胞色素氧化酶（考克斯）是线粒体的末端呼吸复合物，由12-13个亚基组成，其中3个由mtDNA编码，形成酶的催化核心。In S.在酿酒酵母中，复合体的生物发生由大约36个核基因控制。这些编码的一套进口的亚基和各种辅助因子参与表达的催化亚基，膜插入和成熟的亚基2（铜蛋白），血红素A的合成，铜稳态，还有其他事件，目前还不清楚。在过去的资助期间，我们：1）证明了Cox 15 p、铁氧还蛋白和铁氧还蛋白还原酶构成了一个三组分单加氧酶，它能将血红素O转化为血红素A的直接前体，2）鉴定了两个新的基因（COX 18，COX 2 O）负责促进膜插入和亚基2成熟，3）表征了shy 1突变体的抑制子，其表型涉及亚基1（血红素蛋白）作为Shy 1 p作用的靶点，4）与Pierre Rustin博士及其同事合作，证明了一个有致命性神经肌病病史的家族的考克斯缺乏症是由血红素：法呢基转移酶突变引起的。我们的目标是完成血红素A生物合成途径的研究，继续分析shy 1突变体和其余的十几个核基因，其功能在考克斯组装仍然是未知的。在血红素A的生物合成方面，我们希望获得铁氧还蛋白参与血红素O羟基化的更直接的证据，纯化和表征Cox 15 p，并鉴定血红素A的羟基化前体。其次，我们将努力通过脉冲追踪实验和寻找新的抑制剂来研究shy 1突变体中的组装中间体，从而更好地了解shy 1 p的功能。这些研究还应该阐明利氏综合征的生化基础，该综合征已被证明是由SURF 1（SHY 1的人类同源物）突变引起的。与其他COX特异性基因正在进行的功能分析一起，这些研究有望填补我们对这种重要酶如何组装的理解中存在的空白。

DESCRIPTION (provided by applicant): Cytochrome oxidase (COX), the terminal respiratory complex of mitochondria, is composed of 12-13 subunits three of which, encoded by mtDNA, form the catalytic core of the enzyme. In S. cerevisiae, biogenesis of the complex is governed by some 3 dozen nuclear genes. These code for the set of imported subunits and for various accessory factors involved in expression of the catalytic subunits, membrane insertion and maturation of subunit 2 (copper protein), heme A synthesis, copper homeostasis, and still other events that are poorly defined at present. During the past grant period we: 1) obtained evidence that Cox15p, ferredoxin and ferredoxin reductase constitute a three component monooxygenase that converts heme O to the immediate precursor of heme A, 2) identified two new genes (COX18, COX2O) responsible for promoting membrane insertion and maturation of subunit 2, 3) characterized suppressors of shy1 mutants whose phenotype implicate subunit 1 (heme protein) as the target of Shy1p action, 4) collaborated with Dr. Pierre Rustin and his colleagues in demonstrating that the COX deficiency of a family with a history of a fatal neuromyopathy is caused by a mutation in heme:famesyl transferase. Our goals for this coming period is to complete studies on the heme A biosynthetic pathway, to continue the analysis of shy1 mutants and the remaining dozen nuclear genes whose functions in COX assembly remain largely unknown. With respect to heme A biosynthesis, we intend to obtain more direct evidence for the participation of ferredoxin in hydroxylation of heme O, to purify and characterize Cox15p, and to identify the hydroxylated precursor of heme A. Secondly, we will strive to gain better insights into the function of Shy1p by examining the assembly intermediates in shy1 mutants through pulse-chase experiments and by searching for new suppressors. These studies should also shed light on the biochemical basis of Leigh's syndrome, which has been shown to be caused by mutations in SURF1, the human homologue of SHY1. Together with the ongoing functional analysis of other COX-specific genes, these studies are anticipated to fill the existent gaps in our understanding of how this important enzyme is assembled.