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

乙酸细菌的乙醇脱氢酶（ADH）作为乙醇氧化酶呼吸链的初级脱氢酶，在此向泛醌提供电子。ADH是一种膜结合的醌血红蛋白-细胞色素c复合物，由亚基I、II和III组成，含有4个血红素c和吡咯喹啉醌作为假基。除了正常的ADH（活性ADH）外，亚羟酸葡萄糖杆菌还产生非活性ADH，该非活性ADH已被纯化并显示出与活性ADH具有相同的亚基组成和假基。本课题旨在阐明非活性ADH的产生机制和生理功能，并在此过程中获得了以下结果：1)与葡萄糖杆菌一样，醋酸杆菌通过改变培养条件也能产生这种无活性ADH。2)在亚氧化糖葡杆菌中，即使在呼吸底物存在的情况下，静止细胞也会发生无活性ADH向活性ADH的转化，而在球质体中则不会发生。3)利用甲醇醋酸杆菌的II亚基和亚羟葡萄糖杆菌的I/III配合物可制备杂化ADH。通过对天然ADH的动力学性质和氧化还原电位的比较，发现杂交酶中有一个血红素c发生了很大的变化。4)对亚氧氧葡杆菌活性ADH和非活性ADH的氧化还原电位进行了比较，对乙酰乙杆菌中非活性ADH和活性ADH的动力学性质进行了比较，表明4个血红素c中有一个在亚氧氧葡杆菌和乙酰乙杆菌中也发生了变化。5)活性ADHs和非活性ADHs除了具有泛醇还原活性外，还具有泛醇氧化酶活性。无活性ADH的泛醇氧化酶活性也增加。

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.