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

呼吸复合体I通过将NADH氧化与质子转运偶联，在细胞能量代谢中发挥核心作用。它是活性氧物种的主要来源，与衰老有关，其功能障碍被认为与人类神经退行性疾病有关。该复合体由催化电子转移的外围臂和跨膜泵送质子的膜臂组成。这两个过程的耦合尚不清楚。电子进入外围臂末端的络合物，并通过七个铁硫(Fe/S)团簇的链传输到两臂界面的苯醌结合位置。目前还不知道参与将簇组装成复合体的蛋白质。另一个Fe/S团簇不是电子转移链的一部分，但它位于与初级电子受体之间的电子转移距离内。它是严格守恒的，但其功能并不为人所知。链的最远端的簇由一个独特的结合基序协调，该基序包括两个相邻的半胱氨酸残基，这可能使簇能够作为能量转换器运行。缺少这个簇的蛋白质变体与可诱导赖氨酸脱羧酶(CADA)相关，该酶又与参与金属簇组装和修复的蛋白质家族的另外两个蛋白质相互作用。在优先计划的第二阶段，我们将识别与Fe/S簇结合到络合物I中的其他蛋白质，重点是[4Fe4S]簇的结合。将确定其他因素的作用，并将研究它们对硫酰合成酶成熟的重要性。通过使用簇不能被还原的突变体来确定铁/S簇对该络合物的‘非途径’功能。我们认为，该簇调节了进入络合物的电子输入，以响应膜上对苯二酚库的氧化还原状态。我们已经产生了一种变体，其中链的最末端簇由一个规则的结合基序协调。如果团簇的独特连接是能量转换的先决条件，那么该变体应该不能将质子泵过膜。由于其独特的结合基序，这个簇可能需要特殊的帮助才能被包括在复合体中。由于缺少该簇的变体与CADA相关，我们推测CADA参与了该簇的修复或插入。我们将通过电子冷冻显微镜来确定CADA与络合物I的结合。另外两个与CADA相互作用的蛋白质对该复合体组装的贡献将通过在相应的缺失突变体中对该复合体的特征进行研究。我们将从染色体基因和过度生产的质粒中产生复合体，以阐明这些蛋白质的作用。我们认为，这些蛋白使未知的Fe/S簇载体蛋白能够接近簇结合位点。

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.