Structure, biogenesis, and function of mitochondrial complex I

NADH：泛醌氧化还原酶（呼吸复合体I）是一种非常大的膜蛋白复合体，在有氧能量代谢中起中心作用。已知该酶在特定条件下释放有害的氧自由基，例如在心肌梗死后引起组织损伤。线粒体复合体I的功能障碍与许多神经肌肉疾病和神经退行性疾病有关。我们最近以3.6至3.9 Å分辨率解决了有氧酵母脂溶耶氏酵母线粒体复合体I的x射线结构。该结构为解释大型酶复合物中的能量转换和长距离能量转移提供了新的和令人兴奋的复合物I功能。为了全面了解氧化还原连接的质子易位，需要更高分辨率的数据。我们提出了不同的策略来改善结晶，并通过使用抗体片段或T4溶菌酶融合的辅助亚基产生替代晶体形式。目前x射线结构中最有问题的部分位于复合物i的亲水性基质臂的上半部分。我们已经开发了一种可溶外周臂的控制分离和纯化方案，并建议将其结晶为一个离散实体。我们假设泛醌还原位点的协同结构变化触发质子易位。我们建议结晶携带关键残基突变的复合物I变体，以深入了解氧化还原连接质子易位的结构基础。辅助性NUEM亚基属于短链脱氢酶大家族，含有NADPH结合位点。我们的目标是揭示NUEM在线粒体代谢中的未知功能，并解决NUEM与复合物I结合的酰基载体蛋白可能的相互作用。复合体生物发生是一个复杂的多步骤过程。最近，我们分离并研究了一种结合装配因子N7BML （human NDUFAF2）的装配中间体，我们假设N7BML是装配过程中与铁硫簇插入机制相互作用所必需的。我们建议用高分辨率电子显微镜来确定组装中间体的结构。

NADH:ubiquinone oxidoreductase (respiratory complex I) is a very large membrane protein complex with a central function in aerobic energy metabolism. The enzyme is known to release deleterious oxygen radicals under specific conditions causing e.g. tissue damage after myocardial infarction. Dysfunction of mitochondrial complex I is implicated in a number of neuromuscular diseases and neurodegenerative conditions. We have recently solved the X-ray structure of mitochondrial complex I from the aerobic yeast Yarrowia lipolytica at 3.6 to 3.9 Å resolution. The structure offered new and exciting insights into complex I function explaining energy conversion and long range energy transfer in the large enzyme complex. For a comprehensive understanding of redox-linked proton translocation higher resolution data are needed. We propose different strategies to improve crystallization and to generate alternative crystal forms by using antibody fragments or T4 lysozyme fusions of accessory subunits. The most problematic part of the current X-ray structure is located in the upper half of the hydrophilic matrix arm of complex I. We have developed a scheme for controlled separation and purification of the soluble peripheral arm and propose to crystallize it as a discrete entity.We hypothesized that concerted structural changes at the ubiquinone reduction site trigger proton translocation. We propose to crystallize complex I variants carrying mutations of key residues to obtain insight into the structural basis of redox-linked proton translocation. The accessory NUEM subunit of complex I belongs to the large family of short-chain dehydrogenases and contains an NADPH binding site. We aim to unravel the yet unknown function of NUEM in mitochondrial metabolism and to resolve the possible interaction of NUEM with a complex I bound acyl-carrier protein.Complex I biogenesis is a complex multi-step process. Recently, we have isolated and studied an assembly intermediate that binds assembly factor N7BML (human NDUFAF2) and we hypothesized that N7BML is needed to allow interaction with the iron-sulfur cluster insertion machinery during assembly. We propose to determine the structure of the assembly intermediate by high-resolution electron microscopy.