Flemming Dirk, Stolpe Stefan, Schneider Daniel, Hellwig Petra, Friedrich Thorsten
Institut für Organische Chemie und Biochemie, Albert-Ludwigs-Universität Freiburg, Freiburg, Germany.
J Mol Microbiol Biotechnol. 2005;10(2-4):208-22. doi: 10.1159/000091566.
The proton-pumping NADH:ubiquinone oxidoreductase, the respiratory complex I, couples the transfer of electrons from NADH to ubiquinone with the translocation of protons across the membrane. The enzyme mechanism is still unknown due to the lack of a high-resolution structure and its complicated composition. The complex from Escherichia coli is made up of 13 subunits called NuoA through NuoN and contains one FMN and nine iron-sulfur (Fe/S) clusters as redox groups. The pH dependence of the midpoint redox potential of the Fe/S cluster named N2 and its spin-spin interaction with ubiquinone radicals made it an ideal candidate for a key component in redox-driven proton translocation. During the past years we have assigned the subunit localization of cluster N2 to subunit NuoB by site-directed mutagenesis and predicted its ligation by molecular simulation. Redox-induced FT-IR spectroscopy has shown that its redox reaction is accompanied by the protonation and deprotonation of individual amino acid residues. These residues have been identified by site-directed mutagenesis. The enzyme catalytic activity depends on the presence of cluster N2 and is coupled with major conformational changes. From these data a model for redox-induced conformation-driven proton translocation has been derived.
质子泵NADH：泛醌氧化还原酶即呼吸链复合体I，将电子从NADH转移至泛醌的过程与质子跨膜转运相偶联。由于缺乏高分辨率结构及其组成复杂，该酶的作用机制仍不明确。大肠杆菌中的该复合体由13个亚基组成，分别命名为NuoA至NuoN，包含一个FMN和九个铁硫（Fe/S）簇作为氧化还原基团。名为N2的Fe/S簇中点氧化还原电位的pH依赖性及其与泛醌自由基的自旋-自旋相互作用，使其成为氧化还原驱动质子转运关键组分的理想候选者。在过去几年中，我们通过定点诱变确定了簇N2的亚基定位在亚基NuoB上，并通过分子模拟预测了其配位情况。氧化还原诱导的傅里叶变换红外光谱表明，其氧化还原反应伴随着个别氨基酸残基的质子化和去质子化。这些残基已通过定点诱变得以鉴定。该酶的催化活性取决于簇N2的存在，并与主要的构象变化相关联。基于这些数据得出了一个氧化还原诱导的构象驱动质子转运模型。
