Mechanism for Generation and the Physiology of Inactive Form of Alcohol Dehydrogenase in Acetic Acid Bacteria

乙醇脱氢酶（ADH）是乙酸细菌中乙醇氧化酶呼吸链的主要脱氢酶，在呼吸链中为泛醌提供电子。ADH是一种膜结合的醌血红素蛋白-细胞色素c复合物，由亚基I、II和III组成，并含有四个血红素c以及吡咯喹啉醌作为辅基。除了正常的ADH本研究旨在阐明亚氧化葡糖醛酸脱氢酶（ADH）的产生机制和生理功能，并获得以下结果：1）与葡糖醛酸脱氢酶一样，通过改变培养条件，这种无活性的ADH显示在醋杆菌属中产生。2）在弱氧化葡糖酸杆菌中，无活性的ADH向活性的ADH的转化显示甚至在静止细胞中发生，但在原生质球中不发生，3）甲醇醋杆菌亚基Ⅱ和弱氧化葡糖醛酸菌亚基Ⅰ/Ⅲ复合物可形成杂合型ADH。通过比较动力学性质和天然ADH的氧化还原电位，发现杂合酶中的一个血红素c基团发生了很大的变化。4）比较了亚氧化葡糖醛酸菌活性和非活性ADH的氧化还原电位，也比较了醋酸杆菌中非活性ADH和活性ADH的动力学性质，表明4种血红素c中的一种在亚氧化蓝杆菌和醋酸杆菌中的性质也发生了变化。5）活性和非活性ADH除了具有乙醇还原泛醌的活性外，还具有新的功能，即泛醌氧化酶活性。在ADH失活状态下，泛醇氧化酶活性也明显升高。

Alcohol dehydrogenase (ADH) of acetic acid bacteria functions as the primary dehydrogenase of the ethanol oxidase respiratory chain, where it donates electrons to ubiquinone. ADH is a membrane-bound quinohemoprotein-cytochrome c complex which consists of the subunits, I,II and III,and contains four hemes c as well as pyrroloquinoline quinone as prosthetic groups. In addition to normal ADH (active ADH), Gluconobacter suboxydans produces inactive ADH which has been purified and shown to have the same subunit composition and prosthetic groups as active ADH.This research project was performed to elucidate the generation mechanism and also the physiological function of inactive ADH,and the following results have been obtained durint the course.1) As well as Gluconobacter stains, such an inactive ADH was shown to be produced in Acetobacter species by changing the culture conditions.2) In Gluconobacter suboxydans, conversion from inactive to active ADH was shown to occur even in the resting cells, but not in the spheroplast, in the presence of respiratory substrate.3) Hybrid ADH could be prepared from subunit II of Acetobacter methanolicus and subunit I/III complex of Gluconobacter suboxydans. One of the hemes c moieties was shown to be largely changed in the hybrid enzyme by comparing the kinetic properties and the redox potentials of native ADH.4) The redox potentials were compared between active and inactive ADHs of Gluconobacter suboxydans, and also in Acetobacter aceti, the kinetic property of inactive ADH was compared with active ADH,suggesting that one of 4 hemes c is also changed in the property in both Bluconobacter suboxydans and Acetobacter aceti.5) Both active and inactive ADHs were shown to have a novel function, ubiquinol oxidase activity, besides ubiquinone reduction activity with ethanol. The ubiquinol oxidase activity was also shwon to be increased in inactive ADH.