Assembly and Functional Role of the Iron-Sulfur Clusters in Respiratory Complex I

呼吸复合物I通过将NADH氧化偶联至质子转运而在细胞能量代谢中起中心作用。它是活性氧的主要来源，在衰老中起作用，其功能障碍被认为与人类神经退行性疾病有关。该复合物由催化电子转移的外周臂和泵送质子穿过膜的膜臂组成。这两个过程的耦合是未知的。电子在外围臂的尖端进入复合物，并通过七个铁硫（Fe/S）簇合物链被运输到两个臂的界面处的醌结合位点。参与簇组装成复合物的蛋白质尚不清楚。另一个Fe/S簇不是电子转移链的一部分，但它位于电子转移距离的初级电子受体。它是严格保守的，但其功能尚不清楚。链的最远端簇由包含两个相邻半胱氨酸残基的独特结合基序协调，这可能使簇能够作为能量转换器操作。缺乏这个集群的蛋白质变体与诱导型赖氨酸脱羧酶（CadA），这反过来又与其他两个蛋白质的一个蛋白质家族参与组装和修复的金属clusters.In优先级程序的第二个阶段，我们将确定其他蛋白质参与的Fe/S集群的合并到复杂的I，重点是合并的[4Fe 4S]集群。将建立其他因素的作用，并研究其对硫辛酰合酶成熟的重要性。复合物的“非途径”Fe/S簇的功能将通过使用簇不能被还原的突变体来确定。我们建议，这个集群调节电子输入到复杂的响应于氧化还原状态的醌池的膜。我们已经产生了一个变体，其中链的最远端簇由一个规则的结合基序协调。如果簇的独特连接是能量转换的先决条件，则变体应该不能泵送质子穿过膜。由于其独特的结合基序，该簇可能需要特殊的帮助才能被包括在复合物中。由于缺乏该簇的变体与CadA相关，我们推测CadA参与该簇的修复或插入。我们将通过电子冷冻显微镜确定CadA与复合物I的结合。将通过表征相应缺失突变体中的复合物来研究与CadA相互作用的另外两种蛋白质对复合物的组装的贡献。我们将从染色体基因和过量生产质粒中产生复合物，以阐明这些蛋白质的作用。我们建议，这些蛋白质使访问尚未未知的Fe/S簇载体蛋白的簇结合位点。

Respiratory complex I plays a central role in cellular energy metabolism by coupling NADH oxidation to proton translocation. It is a major source of reactive oxygen species, implicating a role in ageing and its dysfunction was suggested to be related to human neurodegenerative diseases. The complex consists of a peripheral arm catalyzing electron transfer and a membrane arm pumping protons across the membrane. The coupling of the two processes is unknown. Electrons enter the complex at the tip of the peripheral arm and are transported to the quinone binding site at the interface of the two arms by a chain of seven iron-sulfur (Fe/S) clusters. Proteins involved in the assembly of the clusters into the complex are not yet known. Another Fe/S cluster is not part of the electron transfer chain, but it is located in electron transfer distance to the primary electron acceptor. It is strictly conserved but its function is not understood. The most distal cluster of the chain is coordinated by a unique binding motif comprising two adjacent cysteine residues, which might enable the cluster to operate as energy converter. A protein variant lacking this cluster is associated with the inducible lysine decarboxylase (CadA), which in turn interacts with two other proteins of a protein family involved in the assembly and repair of metal clusters.In the second period of the priority program, we will identify other proteins involved in the incorporation of Fe/S clusters into complex I with an emphasis on the incorporation of [4Fe4S] clusters. The role of additional factors will be established and their importance for the maturation of lipoyl synthase will be investigated. The function of the ‘off-pathway’ Fe/S cluster for the complex will be determined by using mutants in which the cluster cannot be reduced. We propose that this cluster regulates the electron input into the complex in response to the redox state of the quinone pool of the membrane. We have generated a variant in which the most distal cluster of the chain is coordinated by a regular binding motif. If the unique ligation of the cluster is a prerequisite for energy conversion, the variant should not be able to pump protons across the membrane. This cluster might need special help to be included into the complex due to its unique binding motif. Because a variant lacking this cluster is associated with CadA, we speculate that CadA is involved in the repair or insertion of this cluster. We will determine the binding of CadA to complex I by electron cryo-microscopy. The contribution of two further proteins interacting with CadA to the assembly of the complex will be investigated by characterizing the complex in the corresponding deletion mutants. We will produce the complex from the chromosomal genes and from an overproduction plasmid to clarify the role of these proteins. We propose that these proteins enable the accessibility to the cluster binding site for yet unknown Fe/S cluster carrier proteins.