Abstract
Membrane-bound, pyrroloquinoline quinone-dependent, alcohol dehydrogenase functions as the primary dehydrogenase in the respiratory chain of acetic acid bacteria. In this study, an ability of the enzyme to directly react with ubiquinone was investigated in alcohol dehydrogenases purified from both Acetobacter aceti and Gluconobacter suboxydans by two different approaches. First, it was shown that the enzymes are able to reduce natural ubiquinones, ubiquinone-9or -10, in a detergent solution as well as a soluble short-chain homologue, ubiquinone-1. In order to show the reactivity of the enzyme with natural ubiquinone in a native membrane environment, futhermore, alcohol dehydrogenase was reconstituted into proteoliposomes together with natural ubiquinone and a terminal ubiquinol oxidase. The reconstitution was done by binding the detergent-free dehydrogenase at room temperature to proteoliposomes that had been prepared in advance from a ubiquinol oxidase and phospholipids containing ubiquinone by detergent dialysis using octyl-β-D-glucopyranoside; the enzyme of A. aceti was reconstituted together with ubiquinone-9 and A. aceti cyochrome a1 while G. suboxydans alcohol dehydrogenase was done into proteoliposomes containing ubiquinone-10 and G. suboxydans cytochrome o. The proteoliposomes thus reconstituted had a reasonable level of ethanol oxidase activity, the electron transfer reaction of which was also able to generate a membrane potential. Thus, it has been shown that alcohol dehydrogenase of acetic acid bacteria donates electrons directly to ubiquinone in the cytoplasmic membranes and thus the ethanol oxidase respiratory chain of acetic acid bacteria is constituted of only three membranous respiratory components, alcohol dehydrogenase, ubiquinone, and terminal ubiquinol oxidase.
