Redox-control of the active/deactive transition of mitochondrial complex I

线粒体复合物I是呼吸链中最大和最复杂的酶，与许多退行性疾病有关，是活性氧的主要来源。复合物I可以经历可逆的所谓的活性/失活（A/D）转变。已经表明，仅可接近失活形式的特定半胱氨酸残基的亚硝化防止复合物I的活化，并且可能在某些病理条件下参与阻断呼吸链。我们已经分配这个氧化还原控制复合物I的一个保守的半胱氨酸在natural编码的亚基ND 3。该项目的第一个目的是了解复合物I的活性/失活转变的分子机制，以及它如何通过亚基ND 3中半胱氨酸的修饰来控制。我们将使用我们最近解决的线粒体复合物I的X射线晶体学结构，这表明复合物I的A-和D-形式之间的显着结构变化围绕其半胱氨酸开关，基于分子动力学模拟建立两种状态的结构模型。这些信息将用于指导诱变研究，旨在确定关键参与A/D转换的蛋白质结构域和残基，以了解原子水平上与此过程相关的结构变化。在该项目的第二部分，我们将研究如何根据线粒体的功能状态控制A/D转换和相关的半胱氨酸开关。为此，我们将开发一种定量氧化还原蛋白质组学策略，以解决线粒体和细胞中的A/D过渡态和相关半胱氨酸开关的状态。这将为预测和理解A/D转换和相关半胱氨酸转换机制在健康和疾病中的作用提供有价值的信息。在该项目的第三部分，我们将探讨A/D转换和相关的半胱氨酸开关在线粒体疾病中的作用。为此，我们将利用来自Nijmegen线粒体疾病中心的复杂I相关线粒体疾病患者的大量细胞系。我们对患者成纤维细胞的研究将深入了解与复合物I缺陷相关的病理生理机制中A/D转换的参与，并可能有助于了解线粒体疾病中通常观察到的广泛疾病表型的起源。这也将为健康状态下A/D转换的生理作用提供重要线索。使用我们最近开发的复合体分析方法，我们将搜索多蛋白复合物的线粒体库存调节A/D转换的因素，以达到迄今为止尚未探索的线粒体氧化还原调节机制。

Mitochondrial complex I is the largest and most complicated enzyme of the respiratory chain that has been implicated in numerous degenerative disorders and is a major source of reactive oxygen species. Complex I can undergo a reversible so called active/deactive (A/D) transition. It has been shown that nitrosation of a specific cysteine residue that is accessible only the deactive form prevents activation of complex I and may be involved in blocking the respiratory chain under certain pathological conditions. We have assigned this redox control of complex I to a conserved cysteine in the mitochondrially encoded subunit ND3. The first aim of the proposed project is to understand the molecular mechanism of the active/deactive transition of complex I and how it is controlled by modification of the cysteine in subunit ND3. We will use our recently solved X-ray crystallographic structure of mitochondrial complex I that suggests significant structural changes between the A- and D-from of complex I around its cysteine switch, to build structural models of the two states based on Molecular Dynamics simulations. This information will be used to guide a mutagenesis study aimed at identifying the protein domains and residues critically involved in the A/D transition in order to understand the structural changes associated with this process at the atomic level. In a second part of the project we will study how the A/D transition and the associated cysteine switch is controlled depending on the functional state of mitochondria. For this we will develop a quantitative redox-proteomic strategy to address the A/D transition state and the status of the associated cysteine switch in mitochondria and cells. This will provide valuable information to predict and understand the role of the A/D transition and the associated cysteine switch mechanism in health and disease. In the third part of the project we will explore the role of the A/D transition and the associated cysteine switch in mitochondrial disease. For this, we will take advantage of the large collection of cell lines from patients with complex I related mitochondrial diseases available at the Nijmegen Centre for Mitochondrial Disorders. Our studies with patient fibroblasts will provide insight into the involvement of the A/D transition in pathophysiological mechanisms associated with complex I deficiencies and may contribute to understand the origin of the wide range of disease phenotypes typically observed in mitochondrial disorders. This will also provide important clues for the physiological role of the A/D transition in the healthy state. Using our recently developed complexome profiling approach, we will search the mitochondrial inventory of multiprotein complexes for factors regulating the A/D transition to reach out to so far unexplored mechanisms of mitochondrial redox regulation.