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

线粒体复合体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。复合体I可以经历可逆的所谓的活动/去活动(A/D)转变。已有研究表明，特定半胱氨酸残基的亚硝化反应可防止复合体I的激活，并可能在某些病理条件下参与阻断呼吸链。我们已经将复合物I的这种氧化还原控制分配给线粒体编码的亚基ND3中的一个保守的半胱氨酸。该项目的第一个目标是了解络合物I活性/失活转变的分子机制，以及它是如何通过修饰ND3亚基中的半胱氨酸来控制的。我们将使用我们最近解决的线粒体复合体I的X射线晶体结构，该结构表明复合体I在其半胱氨酸开关周围的A-和D-形式之间发生了显著的结构变化，以分子动力学模拟为基础建立两种状态的结构模型。这些信息将被用来指导一项突变研究，旨在确定在A/D转换中至关重要的蛋白质结构域和残基，以便在原子水平上了解与这一过程相关的结构变化。在项目的第二部分，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A/D转换状态和相关半胱氨酸开关的状态。这将为预测和理解A/D转换和相关的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。在项目的第三部分，我们将探索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，我们将利用奈梅亨线粒体疾病中心提供的复杂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有助于深入了解A/D转变在复杂I缺乏相关的病理生理机制中的参与，并可能有助于理解线粒体疾病中常见的各种疾病表型的起源。这也将为A/D转换在健康状态下的生理作用提供重要线索。使用我们最近开发的复杂组谱方法，我们将在多蛋白复合体的线粒体库存中寻找调节A/D转换的因素，以揭示迄今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.