A Tightly Bound Quinone Functions in the Ubiquinone Reaction Sites of Quinoprotein Alcohol Dehydrogenase of an Acetic Acid Bacterium, Gluconobacter suboxydans

Abstract

Quinoprotein alcohol dehydrogenase (ADH) of acetic acid bacteria is a membrane-bound enzyme that functions as the primary dehydrogenase in the ethanol oxidase respiratory chain. It consists of three subunits and has a pyrroloquinoline quinone (PQQ) in the active site and four heme c moieties as electron transfer mediators. Of these, three heme c sites and a further site have been found to be involved in ubiquinone (Q) reduction and ubiquinol (QH₂) oxidation respectively (Matsushita et al., Biochim. Biophys. Acta, 1409, 154–164 (1999)). In this study, it was found that ADH solubilized and purified with dodecyl maltoside, but not with Triton X-100, had a tightly bound Q, and thus two different ADHs, one having the tightly bound Q (Q-bound ADH) and Q-free ADH, could be obtained. The Q-binding sites of both the ADHs were characterized using specific inhibitors, a substituted phenol PC16 (a Q analog inhibitor) and antimycin A. Based on the inhibition kinetics of Q₂ reductase and ubiquinol-2 (Q₂H₂) oxidase activities, it was suggested that there are one and two PC16-binding sites in Q-bound ADH and Q-free ADH respectively. On the other hand, with antimycin A, only one binding site was found for Q₂ reductase and Q₂H₂ oxidase activities, irrespective of the presence of bound Q. These results suggest that ADH has a high-affinity Q binding site (Q_H) besides low-affinity Q reduction and QH₂ oxidation sites, and that the bound Q in the Q_H site is involved in the electron transfer between heme c moieties and bulk Q or QH₂ in the low-affinity sites.

• ubiquinone
• quinoprotein
• alcohol dehydrogenase
• acetic acid bacteria
• electron transfer in bacterial respiratory chain