Structure, biogenesis, and function of mitochondrial complex I

NADH：泛醌氧化还原酶（呼吸复合物I）是一种非常大的膜蛋白复合物，在有氧能量代谢中具有中心功能。已知该酶在特定条件下释放有害的氧自由基，导致例如心肌梗塞后的组织损伤。线粒体复合物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。我们最近解决了X射线结构的线粒体复合物I从好氧酵母解脂耶氏酵母在3.6至3.9毫米分辨率。该结构提供了新的和令人兴奋的见解复杂的功能，解释能量转换和长距离能量转移的大型酶复合物。为了全面了解氧化还原相关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，以改善结晶和产生替代晶体形式，通过使用抗体片段或T4溶菌酶融合的辅助亚基。目前X射线结构中最有问题的部分位于复合物I的亲水基质臂的上半部分。我们已经开发了一种方案，用于控制分离和纯化的可溶性外周臂，并建议结晶它作为一个离散的实体。我们假设，在泛醌还原位点的协同结构变化触发质子易位。我们建议结晶复杂的I变体携带关键残基的突变，以深入了解氧化还原相关的质子易位的结构基础。复合物I的辅助NUEM亚基属于短链淀粉酶大家族，并且含有NADPH结合位点。我们的目标是解开NUEM在线粒体代谢中的未知功能，并解决NUEM与复合物I结合酰基载体蛋白的可能相互作用。复合物I生物合成是一个复杂的多步骤过程。最近，我们已经分离并研究了结合组装因子N7BML（人NDUFAF2）的组装中间体，并且我们假设在组装期间需要N7BML以允许与铁硫簇插入机器相互作用。我们建议通过高分辨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.