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

醋酸菌的乙醇脱氢酶(ADH)是乙醇氧化酶呼吸链的主要脱氢酶，为泛醌提供电子。ADH是一种膜结合的藜麦血红蛋白-细胞色素c复合体，由亚基I、II和III组成，含有四个血红素c和吡咯喹啉基团作为修复基。除正常的ADH(活性ADH)外，亚氧葡糖杆菌还能产生非活性的ADH，它已被提纯，并且与活性ADH具有相同的亚基组成和辅基。本研究旨在阐明非活性ADH的产生机制和生理功能，并在此过程中得到以下结果：1)和葡萄糖菌菌株，通过改变培养条件，在醋酸菌中可以产生这种非活性的ADH。2)在亚羟基葡糖杆菌中，即使在静息细胞中也可以发生从失活的ADH到活性的ADH的转化，但在球体中则不发生3)甲醇酸乙酸杆菌亚基II和亚基I/III复合体可以制备杂合ADH。通过比较天然ADH的动力学性质和氧化还原电位，发现复合酶中的一种血红素结构发生了很大的变化。4)比较了活性和非活性ADH的氧化还原电位，以及乙酸乙酸杆菌的氧化还原电位，比较了非活性ADH和活性ADH的动力学性质，表明四种血红素c中的一种在亚羟基蓝杆菌和醋酸杆菌中也发生了性质的变化。5)活性和非活性ADH都具有一种新的功能，即泛醌氧化酶活性，除了用乙醇还原泛醌活性外。ADH失活时，泛喹酚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.