Study of cytochrome bo function in Vitreoscilla using a cyo^- knockout mutant

The bacterium, Vitreoscilla, produces a Δμ(Na⁺) across its membrane during respiration. A key enzyme for this function is the cytochrome bo terminal oxidase which, when incorporated into synthetic proteoliposomes, pumps Na⁺ across the membrane upon the addition of a substrate. A Vitreoscilla cytochrome bo knock out (cyo^-) mutant was isolated by transposon mutagenesis using pUT-mini-Tn5Cm. The membranes of this mutant lacked the characteristic 416 nm peak and 432 nm trough in CO difference spectra, which are clearly visible in spectra of the Vitreoscilla wild-type, but peaks at 627, 560, and 530 nm in reduced minus oxidized difference spectra indicate that cytochrome bd is still present. The specific NADH oxidase and ubiquinol-1 oxidase activities of the cyo^- mutant membranes were less than those of Vitreoscilla wild-type and Escherichia coli membranes, and the stimulation of these activities of the mutant and E. coli membranes by 75 mM NaCl was approximately 50% less than that of Vitreoscilla wild-type membranes. The ubiquinol-1 oxidase activity of the cyo^- mutant membranes was inhibited by 10 mM KCN to a lesser degree than that of the Vitreoscilla wild- type and E. coli membranes (50, 80, and 85%, respectively). This result is also consistent with the cyo^- mutant membrane fragments containing only the cytochrome bd terminal oxidase, which is known to be less sensitive to KCN. Although the maximum respiration and growth of the cyo^- mutant were less than those of the wild-type, this mutant is still capable of growing with cytochrome bd alone.