Molecular Mechanism and Structural Basis of Ubiquinone-Redox Reaction in Bacterial Respiratory Chains

本课题在大肠杆菌和亚氧葡萄糖杆菌呼吸链中几种泛素反应酶中寻找泛素结合位点和泛素氧化还原反应的分子机制。目标泛素反应酶为大肠杆菌的细胞色素氧化酶（Cyo）、葡萄糖脱氢酶（GDH）、琥珀酸脱氢酶（SDH）和G.亚羟丹醇脱氢酶（ADH）。为了进行这项研究，主要进行了酶结合醌的检测和酶与叠氮醌的光亲和标记，然后用MALDI-TOF-MS分析标记的亚基和进一步的肽。1)以2-甲基化和3-甲基化醌为前体，成功合成了2-叠氮基q2和3-叠氮基q2，并发现叠氮基部分在紫外照射下被分解。2)建立了2-azido-和3-azido-Q2标记大肠杆菌Cyo的条件。3)大肠杆菌GDH的泛素反应位点存在于C端亲水性PQQ结构域，这可以通过位点诱变制备C端结构域和对泛素的反应性来实现。4)叠氮醌光亲和标记表明，在E. coil SDH中，泛素反应位点存在于膜锚定亚基中。5)用十二烷基麦芽糖苷纯化G. subxydans ADH时，该酶在分子内紧密保留泛素-10，也表现为亚二醌形式。6)在G. subxydans ADH中，亚基II被证明标记为叠氮醌，特别是泛醌还原位点，而不是泛醇氧化位点。标记醌也被发现作为电子介体的泛醌池。

In this research project, search for ubiquinone-binding site and molecular mechanism of redox reaction of ubiquinone was carried out in several ubiquinone-reacting enzymes in the respiratory chains of Escherichia coli and Gluconobacter suboxydans. The objective ubiquinone-reacting enzymes were cytochrome bo oxidase (Cyo), glucose dehydrogenase (GDH), and succinate dehydrogenases (SDH) of E. coli, and alcohol dehydrogenases (ADH) of G. suboxydans. In order to do this research, detection of the bound quinone in the enzymes and also photo-affinity labeling with azidoquinone of the enzymes followed by analysis of the labeled subunit and further peptides by MALDI-TOF-MS were mainly performed.1) 2-azido-Q2 and also 3-azido-Q2 were successfully synthesized via the precursors, 2- and 3- metylated quinone compounds, and also the azido-moiety was found to be decomposed by UV irradiation. 2) Labeling conditions for Cyo of E. coli with both 2-azido- and 3-azido-Q2 were established. 3) Ubiquinone-reacting site of E. coli GDH was shown to be present in the C- terminal hydrophilic PQQ domain, which could be done by preparing the C-terminal domain by site-directed mutagenesis and the reactivity toward ubiquinone. 4) In E. coil SDH, ubiquinone-reacting site was shown to be present in the membrane-anchoring subunit by photo-affinity labeling with azidoquinone. 5) When purified G. suboxydans ADH with dodecyl maltoside, the enzyme retained tightly ubiquinone-10 inside the molecule, and also shown to be as the ubisemiquinone form. 6) In G. suboxydans ADH, the subunit II was shown to be labeled with azidoquinone, especially the ubiquinone reduction site, but not the ubiquinol oxidation site. And also the labeled quinone was found to work as electron mediator to pooled ubiquinone.