Heme trafficking in prokaryotic cytochrome c biogenesis

项目摘要 细胞色素c是高度保守的血红素蛋白，在细胞的电子传递链中发挥作用。 具有呼吸、光合作用和解毒等功能。原核生物生存和繁衍的能力 是电子传输链可塑性的直接结果， 其中细胞色素c是必不可少的成分。人们已经投入了大量的精力来研究 单个细胞色素c，但对它们的生物发生知之甚少，这需要共价 将血红素附着在保守的CXXCH基序上，以实现正确的折叠和功能。尽管它们的多样性，所有的 细胞色素c由三个途径之一产生，系统一(原核生物)、系统二(原核生物)和 系统III(真核生物)，因此，阐明这些途径的分子机制对我们的 了解生物能量学和细胞生存。虽然这三条路径进化的方式不同 完成生物发生的机制，都必须将血红素运输到全细胞色素c合成酶。亚铁血红素是一种 所有生物体中的基本辅因子，不仅在呼吸电子传递链中起作用，而且在 催化、调节和信号传递。然而，我们对血红素运输者和血红素贩运的了解有限。 对血红素的细胞毒性、转运的暂时性和膜研究的技术挑战 蛋白质。因此，我们还必须解决贩运血红素的机制，在这里我们描述我们的长期工作。 阐明血红素传递、运输和依附的一般机制的愿景，从 系统I途径。我们建议：1)鉴定胞质中的血红素受体及其作用机制 交付，2)通过系统I确定血红素运输的路径，以及3)确定周质 亚铁血红素依附。系统I途径由8个完整的膜蛋白(CcmABCDEFGH)和 为研究这些基本的生物学问题提供了一个易于处理的模型系统。Ccmabu建议 通过细菌膜运输血红素并将其连接到CCME上，CCME是周质中的血红素伴侣， 它将血红素运输到全细胞色素c合成酶CcmFH。利用功能性重组大肠杆菌 系统I蛋白用内源性血红素进行纯化，消除了许多技术障碍 与膜蛋白有关。重要的是，血红素附着反应发生在周质中，即 是许多病原体生存所必需的，可能在附着血红素的机制上与 因此，CcmFH合成酶是新型抗微生物药物的潜在靶点。我们的建议 对系统I的研究将同时提供对细胞色素c生物发生和一般 血红素转移的机制，使我们能够研究两个基本的生物过程。一个 这项工作的自然延伸是将学到的一般原则和发展出来的方法应用于其他人 细胞色素c生物发生途径，以及其他原核和真核血红素转运体。

Project Summary Cytochromes c are highly conserved heme proteins that function in electron transport chains for cellular functions such as respiration, photosynthesis and detoxification. The ability of prokaryotes to survive and thrive in diverse, often hostile environments is a direct result of the plasticity of their electron transport chains, of which cytochrome c is an essential component. Much effort has been devoted to studying the roles of individual cytochromes c, but much less is understood about their biogenesis, which requires the covalent attachment of heme at a conserved CXXCH motif for proper folding and function. Despite their diversity, all cytochromes c are made by one of three pathways, System I (prokaryotes), System II (prokaryotes) and System III (eukaryotes), thus elucidation of the molecular mechanisms of these pathways is critical to our understanding of bioenergetics and cellular survival. While the three pathways have evolved different mechanisms to accomplish biogenesis, all must transport heme to a holocytochrome c synthetase. Heme is an essential co-factor in all organisms, functioning not only in electron transport chains for respiration, but also for catalysis, regulation and signaling. Yet our knowledge of heme transporters and heme trafficking is limited due to heme’s cytotoxicity, the transient nature of trafficking and the technical challenges of studying membrane proteins. Thus, we must also address the mechanisms of heme trafficking and here we describe our long-term vision to elucidate the general mechanisms of heme delivery, transport and attachment, beginning with the System I pathway. We propose to 1) identify the cytoplasmic heme receptor and mechanisms of heme delivery, 2) determine the path of heme trafficking by System I, and 3) identify the requirements for periplasmic heme attachment. The System I pathway consists of eight integral membrane proteins (CcmABCDEFGH) and provides a tractable model system to study these fundamental biological questions. CcmABCD are proposed to transport heme across the bacterial membrane and attach it to CcmE, the periplasmic heme chaperone, which trafficks heme to the holocytochrome c synthetase, CcmFH. Utilizing a functional, recombinant E. coli system, the System I proteins purify with endogenous heme, removing many of the technical barriers often associated with membrane proteins. Importantly, the heme attachment reaction occurs in the periplasm, is required for the survival of many pathogens, and likely differs in mechanisms of heme attachment from the eukaryotic synthetase, thus the CcmFH synthetase is a potential target for novel antimicrobials. Our proposed studies on System I will simultaneously provide insights into cytochrome c biogenesis and general mechanisms of heme trafficking, uniquely positioning us to study two fundamental biological processes. A natural extension of this work is to apply the general principles learned and approaches developed to the other cytochrome c biogenesis pathways, as well as to other prokaryotic and eukaryotic heme transporters.