Vladimír Dadák

Effect of oxygen on ubiquinone-10 production by Paracoccus denitrificans

SummaryUbiquinone-10 (Q10) production was measured in batch cultures of Paracoccus denitrificans grown for 8 h at increasing oxygen concentrations (0–21 % O2 in the sparging gas). Whereas the cellular level of Q10 decreased monotonically from 1.2 to 0.5 μmol/g d.w., the total yield of Q10 was maximal at 2.5 % O2 and amounted to 350 nmol (0.3 mg) per L of culture.

The influence of pH on the kinetics of dissimilatory nitrite reduction in Paracoccus denitrificans

Abstract An almost stoichiometric conversion of nitrite to nitrous oxide was observed during the nitrite reduction by Paracoccus denitrificans cells in a medium of pH 6.4. The N 2 O accumulated in the reaction medium and was decomposed only after nitrite had been consumed; when the pH of the medium was higher than 7.3–7.4, nitrous oxide did not accumulate. The activity of N 2 O reductase was, in the whole range of pH 6.4–9.2, higher than the activity of NO − 2 reductase, both activities showing the maximum at the pH higher than 8.0. Using an artificial donor, TMPD plus ascorbate, the maximum activity of NO − 2 reductase, but not N 2 O reductase was shifted by about two pH units to acidic region. The activity of nitrite reductase declined in the presence of N 2 O only at higher pH values. Cytochrome c , as a common electron donor for both N 2 O and NO − 2 reductase, was more oxidized at pH − 2 than in the presence of N 2 O, the opposite being true at pH > 7.3. The increased flux of electrons to cytochrome c has for a constant pH value (6.4) no effect on their distribution over NO − 2 and N 2 O. The results indicate that the distribution of electrons in the terminal part is determined by the different pH optima for NO − 2 reductase and N 2 O reductase, and by a mutual dependence of activities of the two reductases due to the competition for redox equivalents from a substrate.

The role of ubiquinone in linking nitrate reductase and cytochrome o to the respiratory chain of Paracoccus denitrificans

Abstract Three alternatives of the mode of branching in the ubiquinone-cytochrome b region of the anaerobic respiratory chain of Paracoccus denitrificans were experimentally tested. It was found that the view that the constitutive cytochrome b -560 or b -566 serves as an electron donor for the nitrate reductase is incompatible with the proposed scheme of the cyclic electron flow in the bc 1 segment. By means of the extraction procedure, the extent of reduction of ubiquinone was determined in cells utilizing oxygen and nitrate in the presence of antimycin. It was found that the redox response of ubiquinone was consistent with what had been predicted by the pool model of Kroger and Klingenberg, extended for more than one terminal acceptor. Our results are in support of the assumption that in cells of P. denitrificans ubiquinol (QH 2 ) has a function of an electron donor both for nitrate reductase and cytochrome o .

The effect of uncoupler on the distribution of the electron flow between the terminal acceptors oxygen and nitrite in the cells of Paracoccus denitrificans.

The preferential utilization of oxygen, the terminal acceptor, in anaerobically grown cells of Paracoccus denitrificans was abolished in the presence of uncoupler (3 microM carbonyl cyanide m-chlorophenylhydrazone) which brought about a switch to the reduction of nitrite. It has been proved by measuring the redox state of cytochromes that this effect is due to the inhibition of the electron flow to oxygen caused by nitrite, which attains the site of its inhibitory action when the membrane potential is lowered.

The interaction of mucidin with anaerobically grown cells of Paracoccus denitrificans

Abstract Mucidin similar to antimycin inhibits the electron flow to cytochrome c and the enzyme activities dependent on cytochrome c reduction in the cells of Paracoccus denitrificans, but it does not inhibit the electron flow to nitrate reductase and cytochrome o. Unlike antimycin mucidin does not permit a residual electron flow through the cytochrome bc1 region. In the presence of antimycin the electron flow to nitrate is lower than in using mucidin in contrast with a higher extent of cytochrome b reduction. This result is in contradiction to the participation of the constitutive cytochrome b as an electron donor in the nitrate reduction.

Aerobic dissimilatory reduction of nitrite by cells of Paracoccus denitrificans: the role of nitric oxide

Abstract With anaerobically grown cells of Paracoccus denitrificans it was previously found (Kucera, I. and Dadak, V. (1983) Biochem. Biophys. Res. Commun. 117, 252–258) that, in the presence of an uncoupler, nitrite as terminal acceptor was preferred to oxygen, the consumption of which was simultaneously inhibited. In the present study it is shown that besides an increased inhibition of terminal oxidases brought about by NO 2 − anion another potent inhibitor originating in the course of nitrite reductase reaction affects the division of electron flow between oxygen and nitrite. The inhibitor, the creation of which is accompanied by the aerobic nitrite reduction, is formed, even in the absence of an uncoupler, only as a result of a slight inhibition of oxygen respiration exhibited by hydroxylamine addition. From the comparison of the inhibitory effect of the intermediate on aerobically grown cells and membrane vesicles derived from them, it was proved that at neutral pH this substance does not carry an electric charge. A complex absorbing at 563 nm was formed due to the interaction of the inhibitor (generated from nitrite in the presence of uncoupler) with ferricytochrome c from bovine heart. From these findings we were led to conclude that it was most probably nitric oxide (NO).

Function of terminal acceptors in the biosynthesis of denitrification pathway components inParacoccus denitrificans

Limited aeration of cell suspension in growth medium was used to study the kinetics of formation of nitrite reductase and nitrous-oxide reductase and their physiological electron donor, cytochromec-550, during the anaerobic adaptation ofParacoccus denitrificans. The crucial step in the regulation of synthesis of these components is the repressive effect of oxygen while nitrogenous acceptors (NO3−, NO2−, N2O) probably play no role as inducers. The time course of the enzyme activites was analogous (after a lag phase a sharp increase with a maximum after 3 h) and differed from the kinetics of synthesis of cytochromec-550 (gradual rise throughout the 8-h experiment).

Anaerobic adaptation ofParacoccus denitrificans: Sequential formation of denitrification pathway and changes in activity of 5-aminolevulinate synthase and catalase

Aerobically grown cells ofParacoccus denitrificans exhibited a marked increase in the activity of 5-aminolevulinate synthase in the early stage of adaptation to the decreased aeration of the growth medium. The effect was dependent on the functional proteosynthesis; it was not influenced by the presence of nitrate in the medium. An enhancement of nitrate reductase activity could be observed simultaneously; the rise of soluble cytochromec content was somewhat retarded. A significant lag phase of about 1 h appeared in the formation of nitrite reductase, which coincided with the shap drop in the catalase activity of the bacterium.

Respiratory Rate as a Regulatory Factor in the Biosynthesis of the Denitrification Pathway of the Bacterium Paracoccus denitrificans

The decrease in the electron flow of the aerobic respiratory chain of the bacterium Paracoccus denitrificans, owing to either the drop in the saturation of terminal oxidases by oxygen or to the inhibition of the rate of respiration by azide or nitrite, resulted in the synthesis of dissimilatory nitrate reductase and nitrite reductase. The dependence of the resulting activities of the two enzymes (after a three-hour adaptation) on the initial value of the parameter Vmax/kLa (oxidase activity of the volume unit of the culture divided by the volumetric oxygen transfer coefficient) or on the concentrations of the inhibitors had a similar form, characterized by the appearance of a maximum. The increasing parts of the obtained curves reflect the synthesis of enzymes, probably initiated by the increase in the intracellular degree of reduction, the subsequent drop being evidently in connection with the lack of metabolic energy for biosynthesis. The possible mechanisms of the effect of nitrogenous terminal accepto...

Electron transfer from excited tryptophan to cytochrome c: mechanism of phosphorescence quenching?

Parvalbumin, aldolase and liver alcohol dehydrogenase (ADH), proteins exhibiting long-lived phosphorescence lifetimes at room temperature, were examined for their reactivity with ferricytochrome c (cytochrome c Fe3+) as an external electron acceptor. Illumination of a reaction mixture containing protein and cytochrome c in the absence of oxygen brought about reduction of cytochrome c in relation to the duration of light. The largest portion of reduced cytochrome c was found with a sample containing ADH, where a 50% reduction of cytochrome c was reached after 5 min of illumination with a xenon lamp. Parvalbumin and aldolase were about half as effective under the same conditions. Several lines of evidence support the idea that the reaction of cytochrome c occurred by a long-range electron transfer from the excited triplet state of tryptophan. First, cytochrome c quenches the tryptophan phosphorescence and with parvalbumin, its bimolecular quenching rate constant, kq, was 2.9 x 10(6) M-1 s-1. Second, when the illuminated reaction mixture was supplied with 0.2 mM to 1 mM nitrite, a concentration range of nitrite which quenches the tryptophan phosphorescence but not the fluorescence, the amount of reduced cytochrome c on illumination markedly decreased. Finally, for all illuminated protein samples, the extent of cytochrome c reduction occurred parallel to a decrease in tryptophan content as judged from a decrease in fluorescence intensity and/or a decrease in tryptophan absorption at 280 nm.