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

细菌呼吸链中泛醌氧化还原反应的分子机制和结构基础

• 批准号: 12460045
• 负责人: MATSUSHITA Kazunobu
• 金额: $ 9.02万
• 依托单位: Yamaguchi University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 2000
• 资助国家: 日本
• 起止时间: 2000 至 2001
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-12460045/
• 关键词: Azidoquinone; Photo-affinity labeling; Ubiquinone; Escherichia coli Glucose dehydrogenase; Alcohol Dehydrogenase of acetic acid bacteria; Bacterial respiratory chain; 光親和性標識法; 大腸菌チトクロムbo; 呼吸鎖

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.

本研究以大肠杆菌和亚氧化葡糖醛酸菌呼吸链中的几种泛醌反应酶为研究对象，寻找泛醌结合位点和泛醌氧化还原反应的分子机制。目的酶为细胞色素氧化酶（Cyo）、葡萄糖脱氢酶（GDH）和琥珀酸脱氢酶（SDH）。coli的乙醇脱氢酶（ADH）; suboxidans。为了进行这项研究，主要进行了酶中结合醌的检测以及酶的叠氮醌的光亲和标记，然后通过MALDI-TOF-MS分析标记的亚基和进一步的肽。1）通过前体，2-和3-甲基化醌化合物，并且还发现叠氮基部分通过UV照射分解。2)大肠杆菌Cyo的标记条件。大肠杆菌中同时含有2-叠氮基和3-叠氮基-Q2。3)E. coli GDH显示存在于C-末端亲水性PQQ结构域中，这可以通过定点诱变制备C-末端结构域和对泛醌的反应性来完成。4)在大肠coil SDH中，通过叠氮醌的光亲和标记显示，泛素反应位点存在于膜锚定亚基中。5)纯化后G.通过将suboxetans ADH与十二烷基麦芽糖苷进行比较，该酶将泛醌-10紧密地保留在分子内，并且还显示为泛半醌形式。6)In G. subboxerans ADH，亚基II被证明是标记的叠氮醌，尤其是泛醌还原位点，但不是泛醇氧化位点。此外，还发现标记的醌作为电子介质汇集泛醌。