Heme A and cytochrome oxidase biosynthesis

说明(申请人提供)：细胞色素氧化酶(COX)是线粒体的末端呼吸复合体，由12-13个亚基组成，其中3个亚基由mtDNA编码，形成酶的催化核心。在酿酒酵母中，复合体的生物发生由大约30多个核基因控制。这些编码包括一组输入的亚基和各种辅助因子，涉及催化亚基的表达、亚基2(铜蛋白)的膜插入和成熟、血红素A的合成、铜的动态平衡以及目前还不清楚的其他事件。在过去的授权期内，我们：1)获得证据证明Cox15p、铁还蛋白和铁氧还蛋白还原酶构成三种单加氧酶可将血红素O转化为血红素A的直接前体；2)确定了两个新的基因(COX18、COX2O)，它们负责促进膜插入和亚基2的成熟；3)表征了shy1突变体的抑制子，其表型涉及亚单位1(血红素蛋白)作为Shy1p作用的靶点；4)与Pierre Rustin博士和他的同事合作，证明了一个有致命神经病变病史的家庭的COX缺陷是由血红素：甲酰转移酶的突变引起的。我们在未来一段时间的目标是完成对血红素A生物合成途径的研究，继续分析shy1突变和其余十几个核基因，这些基因在COX组装中的功能在很大程度上仍不清楚。在血红素A的生物合成方面，我们打算获得铁氧还蛋白参与血红素O羟化的更直接的证据，纯化和鉴定Cox15p，并鉴定血红素A的羟化前体。其次，我们将通过脉冲追逐实验检测shy1突变体中的组装中间产物，寻找新的抑制因子，从而更好地了解Shy1p的功能。这些研究也应该揭示Leigh综合征的生化基础，已被证明是由SURF1突变引起的，SURF1是SHY1的人类同源物。与正在进行的其他COX特异性基因的功能分析一起，这些研究有望填补我们对这种重要酶是如何组装的理解上的现有空白。

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.