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There was found to be a KCN-insensitive, alternate oxidase chain branching from the ordinary oxidase chain in the respiratory chain of Pseudomonas aeruginosa grown aerobically. The alternate oxidase activity was highly resistant to KCN, and had a lower affinity for oxygen than ordinary cytochrome oxidase did. The branching point of the alternate oxidase chain from the ordinary oxidase chain was shown to be localized behind cytochrome b. The KCN-insensitive alternate oxidase chain was inhibited slightly with antimycin A and intensively with 2-thenoyltrifluoroacetone. The former inhibited the respiration behind cytochrome b and the latter before cytochrome b. N,N,N',N'-Tetramethyl-p-phenylenediamine oxidase-negative mutant (T105) was prepared from P. aeruginosa. The mutant clearly lacked a functional ordinary cytochrome oxidase, but had the KCN-insensitive alternate oxidase chain and could grow aerobically. The KCN-insensitive alternate oxidase chain had a H+/O ratio of 4, suggesting the existence of two energy-coupling sites in the chain. Under the conditions where both ordinary oxidase and alternate oxidase chains were functioning, the H+/O ratio of the parent strain was 5.6. From these data, we also discuss the energetics of the ordinary oxidase chain in the respiratory chain of aerobic P. aeruginosa.

Acinetobacter calcoaceticus is known to contain soluble and membrane-bound quinoprotein D-glucose dehydrogenases, while other oxidative bacteria contain the membrane-bound enzyme exclusively. The two forms of glucose dehydrogenase were believed to be the same enzyme or interconvertible forms. Previously, Matsushita et al. [(1988) FEMS Microbiol. Lett 55, 53-58] showed that the two enzymes are different with respect to enzymatic and immunological properties, as well as molecular weight. In the present study, we purified both enzymes and compared their kinetics, reactivity with ubiquinone homologues, and immunological properties in detail. The purified membrane-bound enzyme had a molecular weight of 83,000, while the soluble form was 55,000. The purified enzymes exhibited totally different enzymatic properties, particularly with respect to reactivity toward ubiquinone homologues. The soluble enzyme reacted with short-chain homologues only, whereas the membrane-bound enzyme reacted with long-chain homologues including ubiquinone 9, the native ubiquinone of the A. calcoaceticus. Furthermore, the two enzymes were distinguished immunochemically; the membrane-bound enzyme did not cross-react with antibody raised against the soluble enzyme, nor did the soluble enzyme cross-react with antibody against the membrane-bound enzyme. Thus, each glucose dehydrogenase is a molecularly distinct entity, and the membrane-bound enzyme only is coupled to the respiratory chain via ubiquinone.

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A membrane-bound D-glucose dehydrogenase [E.C. 1.1.99.a] was solubilized from the membrane of Pseudomonas sp. and purified to a nearly homogeneous state. The solubilized enzyme was monomeric in the presence of 1 % Triton X-100 but aggregated after removing the detergent.The enzyme was a single peptide having a molecular weight of about 90 ,000. The enzyme reacted with various artificial electron acceptors such as phenazine methosulfate, 2,6-dichlorophenolindophenol, Wurster's blue, coenzyme Q1, and ferricyanide . The enzyme had a dual optimum pH depending on the electron acceptor. Reductase activities of the enzyme for 2,6-dichlorophenolindophenol, ferricyanide and coenzyme Q1, were found in more acidic pH region, whereas its activities for phenazine methosulfate and Wurster's blue were observed in more alkaline region. p-Benzoquinone inhibited phenazine metho sulfate reductase activity non-competitively but it inhibited 2,6-dichlorophenolindophenol reductase activity competitively against the acceptor.The enzyme possessed fairly broad substrate specificity, and the reaction product was a gluconolactone.