Heme A and cytochrome oxidase biosynthesis

血红素 A 和细胞色素氧化酶生物合成

• 批准号: 6535483
• 负责人: ALEXANDER A TZAGOLOFF
• 金额: $ 24.73万
• 依托单位: COLUMBIA UNIV NEW YORK MORNINGSIDE
• 依托单位国家: 美国
• 财政年份: 1994
• 资助国家: 美国
• 起止时间: 1994-01-15 至 2006-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6535483
• 关键词: Saccharomyces cerevisiae; copper; crosslink; cytochrome oxidase; enzyme biosynthesis; ferredoxin; functional /structural genomics; fungal genetics; gene expression; gene mutation; genetic screening; heme; hydroxylation; mitochondria; molecular assembly /self assembly; nucleic acid purification; porphyrin biosynthesis; protein purification; protein structure function

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.

描述（申请人提供）：细胞色素氧化酶（COX）是线粒体的末端呼吸复合物，由12-13个亚基组成，其中三个由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 3 打核基因控制。这些编码用于输入亚基集和涉及催化亚基表达、亚基 2（铜蛋白）的膜插入和成熟、血红素 A 合成、铜稳态以及目前尚不清楚的其他事件的各种辅助因子。在过去的资助期内，我们：1）获得了证据，证明 Cox15p、铁氧还蛋白和铁氧还蛋白还原酶构成了三组分单加氧酶，可将血红素 O 转化为血红素 A 的直接前体，2）鉴定了两个新基因（COX18、COX2O），负责促进亚基 2 的膜插入和成熟，3）表征了 shy1 突变体的抑制子，其表型 暗示亚基 1（血红素蛋白）是 Shy1p 作用的靶标，4) 与 Pierre Rustin 博士及其同事合作，证明有致命性神经肌病史的家族的 COX 缺陷是由血红素：法尼基转移酶突变引起的。我们未来一段时间的目标是完成血红素A生物合成途径的研究，继续分析shy1突变体和其余十几个核基因，这些基因在COX组装中的功能仍然很大程度上未知。关于血红素A的生物合成，我们打算获得更直接的证据来证明铁氧还蛋白参与血红素O的羟基化，纯化和表征Cox15p，并鉴定血红素A的羟基化前体。其次，我们将通过脉冲追踪实验和通过检查shy1突变体中的组装中间体来努力更好地了解Shy1p的功能。 寻找新的抑制器。这些研究还应该揭示利氏综合征的生化基础，该综合征已被证明是由 SURF1（SHY1 的人类同源物）突变引起的。结合其他 COX 特异性基因正在进行的功能分析，这些研究预计将填补我们对这种重要酶如何组装的理解中存在的空白。