Cytochrome C Biogenesis

描述（由申请人提供）：细胞色素是大多数生物（包括人类病原体）需氧和厌氧生长所必需的血红素蛋白。相对最近，它已经变得清楚，专门的组装途径和因素是至关重要的细胞色素生物合成。C型细胞色素的组装通过三种途径之一发生，称为系统I，II和III。系统I和II分别有九个和四个组装因子（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。系统III的缺陷导致某些人类遗传疾病。因为只有原核生物，植物和原生动物使用系统I或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至脱辅基细胞色素的血红素，所述半胱氨酸必须被还原以附接至血红素。由于c型细胞色素的组装发生在内膜的外表面，细胞必须将细胞内合成的还原血红素输送到分泌的未折叠脱辅基细胞色素中。在此应用中，我们利用我们以前的成功重组所有三个系统在重组大肠杆菌中研究血红素的体内浓度和功能，是最佳的每个系统。将建立血红素递送途径。此外，将纯化每个系统的最终细胞色素c合成酶（连接酶）活性所需的蛋白质，并表征血红素和脱辅基细胞色素c连接特性、结合和机制。基于我们的研究结果，某些金属卟啉是系统I和II途径的特异性抑制剂，其他卟啉类似物将被连接到脱辅基细胞色素c（或抑制生物发生）的能力进行测试，进一步描绘血红素的性质是至关重要的连接。总之，这项研究将解决哪些蛋白质（S）构成细胞色素c合成酶，血红素是如何交付，什么浓度和血红素的特点是每个系统所需的。研究结果将有助于揭示为什么自然界中出现了三种非常不同的途径以及针对它们的机制。

DESCRIPTION (provided by applicant): Cytochromes are heme proteins essential for the aerobic and anaerobic growth of most organisms, including human pathogens. Relatively recently it has become clear that dedicated assembly pathways and factors are crucial for cytochrome biogenesis. The assembly of c-type cytochromes occurs by one of three pathways, called systems I, II, and III. Systems I and II have nine and four assembly factors (membrane proteins) respectively, while system III uses a single enzyme called cytochrome c heme lyase. Defects in system III result in certain human genetic diseases. Because only prokaryotes, plants, and protozoa use systems I or 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, which must be reduced for attachment to the heme. Since assembly of c-type cytochromes occurs at the outer surface of the inner membrane, cells must deliver reduced heme, synthesized inside the cell, to the secreted unfolded apocytochrome. In this application we take advantage of our previous success for reconstituting all three systems in recombinant Escherichia coli to study the in vivo concentrations and features of heme that are optimal for each system. The heme delivery pathways will be established. Furthermore, the protein(s) required for the final cytochrome c synthetase (ligase) activity of each system will be purified and characterized for heme and apocytochrome c ligation properties, binding, and mechanisms. Based on our findings that certain metal porphyrins are specific inhibitors of the systems I and II pathways, other porphyrin analogs will be tested for their ability to be ligated to apocytochrome c (or to inhibit biogenesis), further delineating the properties of heme that are critical for ligation. In summary, this study will address which protein(s) constitute the cytochrome c synthetases, how heme is delivered, what concentrations and features of heme are required for each system. Results will help unravel why three very different pathways have emerged in nature and mechanisms to target them.