CYTOCHROME C BIOGENESIS

描述(由申请人提供)： 项目摘要/摘要细胞色素是大多数生物体，包括人类病原体有氧和厌氧生长所必需的血红素蛋白。最近，细胞色素生物发生的关键是有专门的组装因子。C型细胞色素的生物发生通过三个途径之一，系统I、II或III。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血红素裂解酶的单一酶。因为只有原核生物、植物和原生动物使用系统I和II，所以这些途径是抗菌剂的潜在靶点。C型细胞色素具有两个半胱氨酸与细胞色素共价连接的血红素。半胱氨酸和亚铁血红素(到Fe2+)必须被还原才能发生附着。此外，所有的细胞色素c都是在内膜外组装并发挥作用的。这项研究考察了系统I和II中的蛋白质如何输送在细胞内合成的血红素，并将其附着到分泌的未折叠的细胞色素c上。该建议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。对于这些纯化的大部分成分，内源血红素已经被捕获，这有助于分析血红素的运输、红牛控制和附着机制。提出了三个目标，第一个目标是系统I，第三个目标是系统II。系统I分两个步骤进行描述。第一步是CcmABCD介导的周质HoloCcmE(氧化态Fe3+状态的血红素)的合成和释放。目的1分析这一步骤，在CCMC和CCME中建立与血红素直接相互作用的残基，以及形成氧化的HoloCcmE的化学机制。HoloCcmE释放步骤与纯化的CcmABCDE蛋白重组。在目标2中分析的步骤2包括CCMF/H介导的holoCcmE还原(到Fe2+)和将这种血红素连接到凋亡素。分析了体内还原的机制(例如，基于苯醌的)，以及CCMF中与CcmH、holoCcmE以及我们发现的一种新的血红素相互作用的残基。目的3分析我们证实的具有血红素输出和细胞色素c合成酶功能的重组系统II CcsBA完整膜蛋白。以下假设将得到检验：CcsBA通过一个“通道”中的两个保守的组氨酸与血红素结合，并在血红素移动到外部的血红素结合部位时保护其免受氧化。我们将在体外研究细胞色素c的外部结合部位和与血红素的结合。原核生物已经进化出数百种不同的细胞色素c，从含有单一血红素的线粒体样细胞色素c到含有十多个血红素分子的特殊c型细胞色素c。结果将揭示血红素转运、血红素红牛控制以及血红素与所有原核生物和植物C型细胞色素附着的机制。

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.