Structure/Function of Complex II Oxidoreductase

项目总结/摘要 线粒体复合物II，也称为琥珀酸脱氢酶（SDH），是一种膜结合的线粒体复合物。 异源四聚体（SDH-ABCD），其四个亚基由核DNA编码。复合物II具有双重功能 通过连接两个基本的能量产生过程。作为TCA循环的一部分， SDHAB结构域将琥珀酸氧化成富马酸。这种氧化产生电子， 通过SDHB亚基的Fe-S簇还原SDHCD膜结构域内的泛醌， 提供能量产生所需的电子传输链的还原当量。作为一个关键的代谢 复合物II的酶功能障碍与使人衰弱的神经变性疾病和肿瘤有关 阵虽然有大量的信息是关于成熟的结构和功能， 复合物II，我们对酶如何组装成功能复合物的理解存在差距。在这 项目中，我们将确定各种组装因子如何共同作用，以插入黄素（FAD）和Fe-S氧化还原 中心到SDHAB亚基，以及如何协调这一过程，以组装一个功能性的复合体II。 目的1：我们最近的进展提供了对组装分子伴侣如何与细菌相互作用的见解。 SdhA亚基形成催化所需的共价FAD键。我们将进一步阐述这些发现， 底物如何使蛋白质结构域运动激活这一组装过程并控制催化作用， 细菌SdhA和人SDHA亚基。这将通过X射线晶体学，SAXS， 和不同中间组装态的双电子电子共振（DEER）谱。 目的2：已知含Fe-S亚基需要特定的组装因子来整合其功能。 集群在这个目标中，我们研究了人SDHB（或细菌SdhB）亚基如何将其三个 独特的Fe-S簇定位于两个独立的蛋白质结构域。我们还将确定是否有独特的 复合物II组装分子伴侣有助于Fe-S簇的插入，或者它们是否稳定Fe-S簇， 蛋白质中间体，用于与细胞Fe-S组装机器的其余部分相互作用。 目的3：复合体II组装故障或组装的复合体在植入过程中分离。 缺血/再灌注可导致线粒体功能的病理。在这个目标中，我们揭示了机制 SDHA和SDHB亚基如何形成功能亚复合物以及与 SDHAB亚复合物的积累。这一目标的一个焦点是组装因子和其他蛋白质 控制可能由SDHAB亚复合物形成的潜在有害的活性氧物质。

PROJECT SUMMARY/ABSTRACT Mitochondrial Complex II, also known as succinate dehydrogenase (SDH) is a membrane-bound heterotetramer (SDH-ABCD) whose four subunits are encoded by nuclear DNA. Complex II has a dual function in the cell by linking two essential energy-producing processes. As part of the TCA cycle the hydrophilic SDHAB domain oxidizes succinate to fumarate. This oxidation generates electrons that are transferred through the Fe-S clusters of the SDHB subunit to reduce ubiquinone within the SDHCD membrane domain to provide reducing equivalent to the electron transport chain needed for energy generation. As a key metabolic enzyme malfunction of Complex II is associated with debilitating neurodegenerative diseases and tumor formation. Although a significant amount of information is available on the structure and function of mature Complex II, there is gap in our understanding of how the enzyme assembles into a functional complex. In this project we will identify how various assembly factors work together to insert the flavin (FAD) and Fe-S redox centers into the SDHAB subunits and how this process is coordinated to assemble a functional Complex II. Aim 1: Our recent progress has provided insight into how assembly chaperones interact with the bacterial SdhA subunit to form the covalent FAD linkage needed for catalysis. We will expand on these findings to show how substrates enable protein domain movements to activate this assembly process and control catalysis in both bacterial SdhA and human SDHA subunits. This will be accomplished by x-ray crystallography, SAXS, and double electron electron resonance (DEER) spectroscopy of different intermediate assembly states. Aim 2: It is known that the Fe-S containing subunit requires specific assembly factors to incorporate its clusters. In this aim we investigate how the human SDHB (or bacterial SdhB) subunit incorporates its three unique Fe-S clusters localized into two separate protein domains. We also will determine whether the unique Complex II assembly chaperones assist with the insertion of the Fe-S clusters, or whether they stabilize the protein intermediates for interaction with the rest of the cellular Fe-S assembly machinery. Aim 3: Either malfunction of Complex II assembly or disassociation of the assembled complex during ischemia/reperfusion can contribute pathologies in mitochondrial function. In this aim we reveal mechanisms of how the SDHA and SDHB subunits form a functional subcomplex and aberrant activities associated with accumulation of the SDHAB subcomplex. A focus of this aim is how assembly factors and other proteins control the potentially deleterious reactive oxygen species that can be formed from the SDHAB subcomplex.