Cultures of Disulfovibrio desulfuricans strain CSN (incubated in a sulfide‐ and sulfate‐free medium) reduced up to 5 mM O2 with H2 as electron donor. Aerobic respiration was not coupled with growth, but resulted in ATP formation. Washed cells incubated in H2‐saturated phosphate buffer revealed respiration rates of up to 250 nmol O2 min−1 mg protein−1. The uncoupler carbonyl‐cyanide m‐chlorophenylhydrazone (CCCP) stimulated the respiration rate and abolished ATP formation. The terminal oxidase has not yet been identified. Respiration was microaerophilic, insensitive to cyanide and azide, but inhibited after heat treatment of the cells (80°C for 10 min). The ph optimum was at pH 6 with less than 50% activity at pH 4.5 and pH 9. Besides H2, organic eletron donors (formate, ethanol, lactate or pyruvate) and inorganic sulfur compounds (H2S, thiosulfate, sulfite) were used as electron donors for aerobic respiration. Sulfite and thiosulfate were oxidized completely to sulfate. The capability of aerobic respiration was also detected in Desulfovibrio vulgaris, D. sulfidismutans, Desulfobacterium autotrophim, Desulfobulbus propionicus, and Desulfococcus multivorans.
Cell extracts of Desulfotomaculum orientis, grown with H2 plus sulfate as sole energy source, revealed hydrogenase activities between 0.3 and 2 μmol H2 per min and mg protein when methyl viologen was used as electron acceptor. With benzyl viologen, methylene blue, FAD or FMN, lower activities were found; NAD was not reduced. The hydrogenase activity was strongly inhibited by CuCl2; however, copper inhibition was not observed with whole cells, indicating that the hydrogenase is located intracellularly. After high‐speed centrifugation of cell‐free extracts, varying proportions, between 11 and 90%, of the hydrogenase were detected in the soluble fraction, the rest being associated with the membrane fraction.
The respiration-driven proton translocation has been measured with the oxygen pulse method and whole cells of the carbon monoxide (CO)-insensitive bacterium Pseudomonas carboxydovorans. -~ H+/O ratios of 5 to 6 were determined with endogenous substrates, pyruvate or hydrogen. In the presence of 100% CO~ H+/O ratios were lowered to about 4. The results indicate that the Co-sensitive electron transport via the cytochromes c and a conserves more energy than the COinsensitive electron transport via the alternative pathway with cytochrome o as terminal oxidase. The CO-sensitive a-type cytochrome was found to translocate 2 protons per oxygen atom reduced, thus accounting for the difference of the energy yield of the CO-sensitive and the CO-insensitive electron transport in P. carboxydovorans. INTRODUCTIONThe CO-oxidizing (carboxydotrophic) bacterium P. carboxydovorans has a branched respiratory chain including a CO-sensitive as well as a CO-insensitive electron pathway [1][2][3]. The CO-sensitive branch contains cytochrome c and an a-type cyto-chrome as terminal oxidase. The CO-insensitive branch, that allows uninhibited growth and respiration in the presence of 90% CO [1], contains an unusual CO-insensitive cytochrome o as terminal oxidase. This cytochrome differed from the CO-insensitive cytochrome o in Rhodopseudomonas capsulata [4] in that it revealed the lowest midpoint potential ( -100 mV) of all cytochromes present in the cells. Its function as terminal oxidase is made possible by a very high affinity to oxygen (Km < 0.05 ~M 02, unpublished results). It was therefore of interest to study whether electron transport via this unusual cytochrome oxidase yields the same energy as electron transport via the CO-sensitive pathway.P. carboxydovorans can grow autotrophically with H 2 as well as with CO as electron donor. Both substrates deliver electrons to the same site of the respiratory chain [3]. However, cells growing with H 2 have a higher growth rate (t a = 10 h) than cells growing with CO (t d = 20 h). We suspected that in the presence of CO the CO-sensitive pathway via the cytochromes c and a is blocked and that the lower growth rate might be due to a lower energy yield of the CO-insensitive electron pathway. The proton-translocation measurements described in this study confirm this view and indicate that the difference of the energy yield is 0378-1097/84/$03.00
The midpoint redox potentials (E′0) of the cytochromes of Pseudomonas carboxydovorans have been studied by means of coupled spectrum deconvolution and potentiometric analysis. Membranes of cells grown on different substrates (CO; H2+ CO2; or pyruvate) contained cytochromes with similar absorption peaks and redox potentials. The cytochromes of the CO‐sensitive main electron pathway of the respiratory chain revealed redox potentials in the same range as mitochondrial cytochromes (cytochrome b‐555, about −20 mV; cytochrome c and cytochrome a, about +220 mV). For the cytochromes of the CO‐insensitive alternative electron pathway, which allows uninhibited growth and respiration in the presence of high concentrations of CO, redox potentials of approx. +50 mV (cytochrome b‐558) and −11 to −215 mV (cytochrome b‐561) were determined. Cytochrome [ib‐561], earlier proposed as the alternative terminal oxidase o in this organism, was shown to possess the lowest half reduction potential of all the cytochromes present in the cells. Measurements of the apparent Km value for oxygen revealed a low affinity of cytochrome a (Km/ 5 υ M O2) and a very high affinity of the CO‐insensitive oxidase (Km < 0.5 μ M O2). The high affinity to oxygen might be responsible for the CO‐insensitivity of this unusual cytochrome o.
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