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/摘要细胞色素是血红素蛋白，对于大多数生物体（包括人类病原体）的需氧和厌氧生长至关重要。最近，人们已经清楚，专用的组装因子对于细胞色素的生物合成至关重要。 c 型细胞色素的生物合成通过系统 I、II 或 III 三种途径之一发生。系统 I 有八个 (CcmABCDEFGH)，系统 II 有两个 (CcsBA) 专用组装因子（膜蛋白），而系统 III 使用一种称为细胞色素 c 血红素裂解酶的酶。由于只有原核生物、植物和原生动物使用系统 I 和 II，因此这些途径代表了抗菌药物的潜在靶点。 c型细胞色素具有与脱辅基细胞色素在两个半胱氨酸处共价连接的血红素。半胱氨酸和血红素（至 Fe2+）必须被还原才能发生附着。此外，所有细胞色素 c 在内膜外组装并发挥作用。这项研究探讨了系统 I 和 II 中的蛋白质如何传递细胞内合成的血红素，并将其附着到分泌的未折叠脱辅基细胞色素 c 上。该提案利用了我们之前从重组大肠杆菌中纯化系统 I 和 II 的所有蛋白质的成功经验。对于大多数这些纯化成分，内源血红素已被捕获，有助于血红素运输、氧化还原控制和附着机制的分析。提出了三个目标，前两个目标针对系统 I，第三个目标针对系统 II。系统 I 分两步描述。步骤 1 是 CcmABCD 介导的周质 HoloCcmE（氧化 Fe3+ 状态的血红素）的合成和释放。目标 1 分析此步骤，确定 CcmC 和 CcmE 中直接与血红素相互作用的残基以及形成氧化 HoloCcmE 的化学机制。 HoloCcmE 释放步骤用纯化的 CcmABCDE 蛋白重建。目标 2 中分析的步骤 2 包括 CcmF/H 介导的 holoCcmE 还原（至 Fe2+）以及该血红素与脱辅基细胞色素的连接。分析了体内还原机制（例如基于醌的还原），以及 CcmF 中与 CcmH、holoCcmE 和我们发现的新型 b 血红素相互作用的残基。目标 3 分析重组系统 II CcsBA 整合膜蛋白，我们证明该蛋白具有血红素输出和细胞色素 c 合成酶功能。将测试以下假设：CcsBA 通过“通道”中的两个保守组氨酸结合血红素，并在血红素移动到外部血红素结合位点时保护血红素免受氧化。将在体外研究外部脱辅基细胞色素 c 结合位点和与血红素的附着。原核生物已经进化出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附着在单个多肽上的非凡的 c 型细胞色素。这里的结果将揭示血红素运输、血红素氧化还原控制以及血红素与所有原核和植物 c 型细胞色素的附着的潜在机制。