CYTOCHROME C BIOGENESIS

细胞色素 C 生物合成

• 批准号: 8715815
• 负责人: Robert G. Kranz
• 金额: $ 34.2万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1994
• 资助国家: 美国
• 起止时间: 1994-09-01 至 2015-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8715815
• 关键词: Address; Aerobic; Binding; Binding Sites; Bioenergetics; Biogenesis; Biological Assay; Cell membrane; Cells; Chemicals; Chloroplasts; Complex; Cysteine; Cytochrome c Group; Cytochromes; Electron Transport; Engineering; Enzymes; Escherichia coli; Eukaryota; Figs - dietary; Growth; Heme; Hemeproteins; Histidine; Human; Hydroquinones; In Vitro; Integral Membrane Protein; Intracellular Transport; Ligands; Ligase; Ligation; Mediating; Membrane; Membrane Proteins; Mitochondria; Modeling; Molecular; One-Step dentin bonding system; Organism; Oxidoreductase; Pathway interactions; Plants; Prokaryotic Cells; Protein C; Proteins; Protozoa; Quinones; Recombinants; System; Testing; Transmembrane Domain; abstracting; adduct; antimicrobial drug; base; cofactor; cytochrome C synthetase; cytochrome c; frontier; in vivo; novel; oxidation; pathogen; periplasm; polypeptide; reconstitution; success

DESCRIPTION (provided by applicant): Project Summary/Abstract Cytochromes are heme proteins essential for the aerobic and anaerobic growth of most organisms, including human pathogens. Recently it has become clear that dedicated assembly factors are crucial for cytochrome biogenesis. The biogenesis of c-type cytochromes occurs by one of three pathways, systems I, II, or III. System I has eight (CcmABCDEFGH) and system II has two (CcsBA) dedicated assembly factors (membrane proteins), while system III uses a single enzyme called cytochrome c heme lyase. Because only prokaryotes, plants, and protozoa use systems I and II, these pathways represent potential targets for antimicrobial agents. The c-type cytochromes possess heme that is covalently ligated to the apocytochrome at two cysteines. The cysteines and heme (to Fe2+) must be reduced for attachment to occur. Moreover, all cytochromes c are assembled and function outside of the inner membrane. This study examines how proteins in systems I and II deliver heme, synthesized inside the cell, and attach it to the secreted unfolded apocytochrome c. The proposal takes advantage of our previous success in purifying all proteins of systems I and II from recombinant Escherichia coli. For most of these purified components, endogenous heme has been trapped, facilitating analyses of heme transport, red-ox control, and attachment mechanisms. Three aims are proposed, the first two for system I, and the third for system II. System I is described in two steps. Step 1 is the CcmABCD-mediated synthesis and release of periplasmic holoCcmE (heme in the oxidized Fe3+ state). Aim 1 analyzes this step, establishing residues in CcmC and CcmE that directly interact with heme and the chemical mechanisms to form the oxidized holoCcmE. The holoCcmE release step is reconstituted with purified CcmABCDE proteins. Step 2, analyzed in Aim 2, includes the CcmF/H-mediated reduction of holoCcmE (to Fe2+) and ligation of this heme to apocytochrome. The mechanism of reduction in vivo (e.g., quinone-based) is analyzed, as well as residues in CcmF for interacting with CcmH, holoCcmE, and a novel b heme we discovered. Aim 3 analyzes the recombinant system II CcsBA integral membrane protein that we demonstrated has heme export and cytochrome c synthetase functions. The following hypothesis will be tested: CcsBA binds heme via two conserved histidines in a "channel" and protects the heme from oxidation as it moves to an external heme binding site. The external apocytochrome c binding site and attachment to heme will be studied in vitro. Prokaryotes have evolved hundreds of different cytochromes c, ranging from the mitochondrial-like cytochrome c with a single heme to extraordinary c-type cytochromes with over ten heme molecules attached to a single polypeptide. Results here will unravel the mechanisms underlying heme transport, heme red-ox control, and heme attachment to all prokaryotic and plant c-type cytochromes.

描述（由申请人提供）： 项目摘要/摘要细胞色素是大多数生物（包括人病原体）的有氧和厌氧生长所必需的血红素蛋白。最近，很明显，专用组装因子对于细胞色素生物发生至关重要。 C型细胞色素的生物发生由三个途径之一，系统I，II或III发生。系统I具有八个（CCMABCDEFGH），系统II具有两个（CCSBA）专用的装配因子（膜蛋白），而系统III使用一种称为细胞色素C Heme裂解酶的单个酶。因为只有原核生物，植物和原生动物使用系统I和II，所以这些途径代表了抗菌剂的潜在靶标。 C型细胞色素具有血红素，该血红素在两个半胱氨酸处共价绑扎在启示色。必须减少半胱氨酸和血红素（至Fe2+）以进行附着。此外，所有细胞色素c均组装并在内膜外部功能。这项研究研究了系统I和II中的蛋白质如何在细胞内部合成血红素，并将其连接到分泌的展开的旋转式染色体c。该提案利用了我们先前从重组大肠杆菌中净化系统I和II的所有蛋白质的成功。对于大多数这些纯化的组件，内源性血红素已被困，促进了血红素转运，红色氧化和固定机制的分析。提出了三个目标，第一个针对系统I，第三个针对系统II。系统I分为两个步骤。步骤1是CCMABCD介导的合成和周质全蛋白的释放（氧化Fe3+状态中的血红素）。 AIM 1分析此步骤，建立在CCMC和CCME中的残基，这些残基与血红素直接相互作用以及化学机制形成了氧化的Holoccme。 Holoccme释放步骤用纯化的CCMABCDE蛋白重组。在AIM 2中分析的步骤2包括CCMF/H介导的Holoccme（至Fe2+）的降低以及该血红素与启示色素的连接。分析了体内还原（例如，基于喹酮）的机制，以及CCMF中与CCMH，Holoccme相互作用和我们发现的新型B血红素的残基。 AIM 3分析了我们证明的重组系统II CCSBA积分膜蛋白具有血红素出口和细胞色素C合成酶功能。将测试以下假设：CCSBA通过“通道”中的两个保守的组氨酸结合血红素，并在移动到外部血红素结合位点时保护血红素免受氧化的影响。将在体外研究外部旋转色素C结合位点和对血红素的附着。原核生物已经进化了数百种不同的细胞色素C，范围从具有单个血红素的线粒体样细胞色素c到具有十多个血红素分子的非凡C型细胞色素。此处的结果将揭示血红素转运，血红素红氧控制和血红素附着在所有原核和植物C型细胞色素上的机制。