CYTOCHROME C BIOGENESIS

描述（由申请人提供）： 细胞色素是大多数生物体（包括人类病原体）需氧和厌氧生长所必需的血红素蛋白。最近，它已成为明确的，专门的组装因子是至关重要的细胞色素的生物合成。C型细胞色素的生物发生通过三种途径之一，系统I，II或III。系统I有八个（CcmABCDEFGH）和系统II有两个（CcsBA）专用的组装因子（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。因为只有原核生物，植物和原生动物使用系统I和II，这些途径代表了抗菌剂的潜在目标。C型细胞色素具有在两个半胱氨酸处共价连接到脱辅基细胞色素的血红素。半胱氨酸和血红素（还原为Fe 2+）必须被还原才能发生附着。此外，所有细胞色素c都在内膜外组装和发挥作用。本研究探讨了系统I和II中的蛋白质如何传递细胞内合成的血红素，并将其附着在分泌的未折叠脱辅基细胞色素c上。该提案利用了我们以前在纯化重组大肠杆菌的系统I和II的所有蛋白质方面的成功。对于大多数这些纯化的成分，内源性血红素已被捕获，促进血红素运输，红牛控制和附着机制的分析。提出了三个目标，前两个为系统I，第三个为系统II。系统I分两步描述。第1步是CcmABCD介导的周质holoCcmE（氧化态Fe 3+的血红素）的合成和释放。目的1分析这一步骤，建立CcmC和CcmE中直接与血红素相互作用的残基以及形成氧化的holoCcmE的化学机制。用纯化的CcmABCDE蛋白重建holoCcmE释放步骤。第2步，在目标2中分析，包括CcmF/H介导的holoCcmE还原（至Fe 2+）和该血红素与脱辅基细胞色素的连接。体内还原机制（例如，醌基），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。目的3分析重组系统II CcsBA整合膜蛋白，我们证明有血红素输出和细胞色素c合成酶的功能。将检验以下假设：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，从具有单个血红素的类细胞色素c到具有超过10个血红素分子连接到单个多肽的非凡的c型细胞色素。本研究结果将揭示血红素转运、血红素氧化还原控制以及血红素附着于所有原核生物和植物c型细胞色素的机制。

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.