Electron transfer in Paracoccus denitrificans with the modified fbc operon.

Membrane fragments of two mutant strains of Paracoccus denitrificans genetically modified in the bc(1) complex have been studied for comparison of enzymic activities of succinate-cytochrome-c reductase and its components, viz. succinate dehydrogenase (Complex II) and ubiquinol-cytochrome-c reductase (Complex III) and their response to changes in concentration of succinate, cytochrome c, ionic strength, pH, temperature and sensitivity to antimycin A. The mutants synthesized and assembled the b and c hemes in the ratio characteristic for the wild type strain. The mutant strain M 71 expressing the truncated copy of cytochrome c(1) (devoid of a stretch of 150 mainly acidic amino acids) was less sensitive to increasing concentration of cytochrome c and changes in ionic strength of the medium, but maintained the original affinity to succinate and sensitivity to antimycin A. The mutant strain M 36 with an overexpressed bc(1) content showed the highest response to changes in ionic strength and physical parameters, exhibited the lowest turnover number values with succinate-cytochrome-c reductase, but positively affected the succinate dehydrogenase. In view of the interaction of the redox components in native membranes the functional analyses of separated Complexes II and III should be regarded with caution.

Electrostatic attraction between cytochrome bc1 and cytochrome c affects kinetics of cytochrome c reduction.

The kinetics of the ubiquinol-cytochrome c reductase reaction was examined using membrane fragments and purified bc(1) complexes derived from a wild-type (WT) and a newly constructed mutant (MUT) strains of Paracoccus denitrificans. The cytochrome c(1) of the WT samples possessed an additional stretch of acidic amino acids, which was lacking in the mutant. The reaction was followed with positively charged mitochondrial and negatively charged bacterial cytochromes c, and specific activities, apparent k(cat) values, and first-order rate constant values were compared. These values were distinctly lower for the MUT fractions using mitochondrial cytochrome c but differed only slightly with the bacterial species. The MUT preparations were less sensitive to changes of ionic strength of the reaction media and showed pure first-order kinetics with both samples of cytochrome c. The reaction of the WT enzyme was first order only with bacterial cytochrome c but proceeded with a non-linear profile with mitochondrial cytochrome c. The analysis of the reaction pattern revealed a rapid onset of the reaction with a successively declining rate. Experiments performed in the absence of an electron donor indicated that electrostatic attraction could directly participate in cytochrome c reduction.

Evidence of complex formation between alkaline phosphatase and a pro-apoptotic hemoprotein cytochrome c.

Two proteins, alkaline phosphatase (AP) and cytochrome c (cyt c) which seem to be involved in the apoptotic cell death program were examined on their interaction. Intestinal AP affects ferricytochrome c (cyt c(FeIII)) by changing its optical properties, redox state and conformation. The effect proceeded over the course of hours with a gradual decrease in free cyt c(FeIII) as the AP concentration increased. A heme containing high molecular species was created in the first stage of interaction of the proteins in neutral, acidic (pH 2.6), alkaline (pH 8.3), low ionic strength (10 mmol/l phosphate), and high ionic strength (0.5 mol/l NaCl) media. Further complexation was favored by higher pH values and temperature. Differential scanning calorimetry revealed a decrease in enthalpy of the thermodenaturation temperature (Tm) of cyt c at 84.5 degrees C due to the AP addition. Increments of AP in the mixtures resulted in the appearance of Tm peaks at 68 degrees C and 61 degrees C. Electrophoretic analysis of the commercial samples of intestinal APs showed main fractions from 63.2 kDa to 72.9 kDa and from 172.9 up to 179.0 kDa. Changes in positions and intensities of the bands were detected upon longer incubation (24 h) with cyt c. The electrophoretic pattern of the bacterial AP was homogeneous with one fraction of 43.7 kDa showing no alteration due to the cyt c presence. Gel permeation chromatography of incubated mixtures of intestinal APs and cyt c confirmed the creation of new heme containing complexes.

Interaction of alkaline phosphatase with cytochrome c.

Alkaline phosphatase (AP) a protein which exhibits long-lived phosphorescence lifetime and ferricytochrome c as a phosphorescence quenching agent were examined. The excitation of the tryptophan triplet state resulted in cytochrome c reduction confirming long-range electron transfer as the quenching mechanism. The rate of electron transfer was not related to the length of the illumination interval; an additional reaction between the two proteins leading to cytochrome c reduction was detected. The reaction which proceeded in the dark was not sensitive to oxygen, was dependent on pH, and on the AP to cytochrome c ratio. At optimum 68 +/- 4% of the total cytochrome c could be reduced due to the presence of AP. On incubation of the two proteins the conformation of cytochrome c was altered as was evidenced by its decreased reducibility by ascorbate, by the disappearance of the absorption band at 695 nm, by the appearance of the new band at 620-640 nm, and by a change in circular dichroism spectra witnessing a structural alteration in the vicinity of the heme cleft. This was characterized by a profound increase in positive elipticity at 400 nm and by a reversible change in the magnitude of negative elipticity at 417 nm. The reaction was not significantly affected by the addition of sulfhydryl-binding and metal-complexing agents.

Interaction of carbonyl cyanide 3-chlorophenylhydrazone with cytochrome c oxidase.

Cytochrome c oxidase binds protonophore carbonyl cyanide 3-chlorophenylhydrazone (CCCP) with high affinity. There are 1.46 high-affinity binding sites per cytochrome c oxidase for CCCP with dissociation constant 2.7 x 10(-7) mol/l. The bond between the CCCP and cytochrome c oxidase accomplishes through the group on cytochrome c oxidase with pKa 6.64 and is based on the electrostatic interaction. Interaction of CCCP with low-affinity binding sites of cytochrome c oxidase induces the shift of the anion CCCP spectrum to UV-region. The similar effect is characteristic for CCCP interaction with protons. Lipophilic non-dissociated derivative NCH3CCP is not binding to cytochrome c oxidase.

Modified simplified method for isolation of lysostaphin from the culture filtrate of Staphylococcus staphylolyticus.

The present paper reports a modified method for isolation of lysostaphin--a bacteriolytic agent with specific affinity for staphylococcal cell wall. The proposed purification scheme includes three steps. The first procedure is ultrafiltration through a membrane filter giving a yield of 75.6%. The result of ultrafiltration is a concentrated, 10-times purified preparation of lysostaphin with specific activity 0.62 U/mg which can be used for digestion of S. aureus cells. Further step, performed by ion-exchange chromatography on DEAE-cellulose, yields a 60-times purified preparation containing a mixture of enzyme components of lysostaphin. The yield of this step is 47.2%, the preparation contains 3.54 U/mg protein. Using gel filtration on Sephadex G-50 a component with hexosaminidase activity was separated from the endopeptidase component on the basis of molar mass difference. A 270-times purified preparation of lysostaphin-endopeptidase with minimum of contaminating substances was obtained in this step. The yield of gel filtration was 22.1%, specific activity increased up to 16.3 U/mg protein.

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.

Separate binding sites for antimycin and mucidin in the respiratory chain of the bacterium Paracoccus denitrificans and their occurrence in other denitrificans bacteria.

By means of the method of fluorimetric titration it has been shown that mucidin does not affect the attachment of antimycin to membranes from anaerobically grown Paracoccus denitrificans. The fluorimetric titration with antimycin can be used in the determination of the amount of the cytochrome bc1 complex in the membrane. In cells inhibited with antimycin, the oxidation of cytochromes c was accompanied by the reduction of cytochrome b; in the presence of mucidin this effect did not take place. The results, which indicated a difference in binding sites, were interpreted in terms of the Q-cycle [Mitchell (1976) J. Theor. Biol. 62, 327-367; Trumpower (1981) Biochim. Biophys. Acta 639, 129-155]. Comparable sensitivity towards antimycin and mucidin was shown by other typical denitrifying bacteria: Pseudomonas stutzeri and Alcaligenes xylosoidans, subspecies denitrificans.

Control of respiration rate in non-growing cells of Paracoccus denitrificans.

By means of fluorimetric measurement and by direct determination of intracellular NAD+ and NADH contents, it was proved that the respiration rate of Paracoccus denitrificans cells utilizing glucose is limited by processes preceding NADH oxidation in the respiratory chain, so that the membrane NADH dehydrogenase is not saturated by its substrate. In the separated membrane fraction on saturation with exogenous NADH the main limiting factor is represented by NADH: ubiquinone oxidoreductase.

Is the ubiquinone pool in the respiratory chain of the bacterium Paracoccus denitrificans really unhomogeneous?

We have established the participation of a mobile redox pool in the respiratory chain of anaerobically grown bacterium Paracoccus denitrificans. In testing the kinetical homogeneity of the pool it was found that the ratio of fluxes of electron transport toward the terminal acceptors oxygen and nitrate was coincident for the respiratory substrates NADH and succinate; this provides evidence against the preferential link of one dehydrogenase with a distinct terminal enzyme through the separate pool of ubiquinone. The deviation from the expected behavior observed in comparing the titration of NADH oxidase and succinate oxidase with respiratory inhibitors such as mucidin (inhibitor in the bc1 region) or cyanide can be accounted for by the activation of succinate dehydrogenase upon the increase in the reduced state of respiratory components during the titration.

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 function of cytoplasmic membrane of Paracoccus denitrificans in controlling the rate of reduction of terminal acceptors.

The rate of reduction of terminal acceptors (nitrate, nitrite, and oxygen) in anaerobically grown cells of Paracoccus denitrificans increased on permeabilization of cytoplasmic membrane. It was proved that under aerobic conditions the increase of the rate of nitrate reduction was caused by: (i) the abolishment of the permeability barrier for nitrate, (ii) the enhancement of the influx of redox equivalents to the respiratory chain due to the stimulation of succinate dehydrogenase reaction, and (iii) the inhibition of electron flow to oxygen by endogenously formed nitrite. Nitrite inhibits oxygen reduction by its interaction with the terminal part of the respiratory chain (I50 = 15 microM) localized at the inner aspect of the cytoplasmic membrane. The distribution of nitrite between intact cells and the suspension medium follows the Nernst equation for monovalent anion. The possible physiological consequences of the low intracellular nitrite concentration are discussed.

Berberine derivatives as cationic fluorescent probes for the investigation of the energized state of mitochondria.

The interaction of rat liver and bovine heart mitochondria with a series of fluorescent, cationic berberine derivatives varying in the length of alkyl chain has been investigated. An increase in the hydrophobicity of the derivative was accompanied by a larger value of the partition coefficient and by binding to a more hydrophobic region of the inner mitochondrial membrane. It was found that berberines could be used as sensitive indicators of processes which take place on the outer surface of the mitochondrial membrane; the greatest (15-fold) increase in fluorescence was obtained with 13-methylberberine in the energized state of mitochondria. The fluorescence increase was due to the increase in fluorescence quantum yield although a small increase in the amount of bound derivative could also be detected upon energization. The fluorescence was linearly dependent on the magnitude of the membrane potential. In parallel with an observed fluorescence enhancement a considerable decrease in rotational mobility was found. We suggest that berberines move in the inner membrane according to the polarity of the membrane potential; consequently, deeper immersion in the less polar region in the energized state brings about a larger fluorescence increase. More hydrophobic derivatives inhibited NAD-linked respiration in rat liver mitochondria but exerted no effect on succinate oxidation up to 10 microM concentration.

Phosphate transport system in paracoccus denitrificans.

Pi uptake in cells or spheroplasts of Paracoccus denitrificans is biphasic; only the first rapid phase represents net Pi transport. The second phase is limited by the rate of Pi utilization inside the cell, i.e., mainly by its esterification, and as such it was inhibited by DCCD. The Pi/dicarboxylate antiporter does not seem to be operative, and its inhibitor n-butylmalonate did not exert specific inhibition. Pi transport is inhibited by SH reagents; the most potent inhibitor is PCMB, and mersalyl is much less effective. However, neither inhibitor affects efflux of accumulated Pi. The gradient of potassium ions may be involved in the Pi uptake, which is lowered in the presence of valinomycin. FCCP alone does not release accumulated Pi from spheroplasts unless they are preincubated with SCN-. The results indicate that Pi enters the cell by symport with protons.

Changes in the cytochrome c content during the aerobic adaptation of Paracoccus denitrificans.

Concentration of cytochrome c decreases when shaking anaerobically grown cells in a non-growth medium down to 50% of the original amount in the cell, depending on the degree of aeration. Only 10-20% of this amount can be found in the adaptation medium. The main portion of cytochrome c is degraded during the adaptation. Inhibitors of proteinases do not influence the degradation. Addition of mammalian cytochrome c fully prevents the degradation of bacterial cytochrome c but not its release from cells. Potassium hexacyanoferrate(III) exhibits a similar effect as oxygen. The degradation system is probably localized in the periplasmic space of the cells.

Tetraphenylborate-sensitive electrode for measuring membrane potential.

The paper describes the construction of a new type of ion-selective electrode sensitive to tetraphenylborate (TPB-) and its electric characteristics. The electrode responds to increasing concentrations of the TPB- anion in accordance with the Nernst equation and can be used down to 0.1 microM concentration. The applicability of the electrode for measuring the membrane potential (positive inside) was proved in inside-out oriented membrane vesicles derived from Paracoccus denitrificans. The calculated values were 175 +/- 12 mV with NADH and 180 +/- 6 mV with succinate.

Interaction of lipophilic quinones with membrane fragments of Paracoccus denitrificans and Staphylococcus epidermidis.

In quinone-depleted mitochondrial and Paracoccus denitrificans membranes the quantum yield of fluorescence of ostruthin (6-geranyl-7-hydroxycoumarin) was maintained, whereas an increase in the quantum yield took place after extraction of Staphylococcus epidermidis membrane. A marked quenching effect of ubiquinone and menaquinone each with two isoprene units in the side chain on the ostruthin fluorescence was found with all types of quinone-depleted particles. When the homogues of menaquinone and ubiquinone with six isoprene units in the side chain were re-incorporated, a quenching of the ostruthin fluorescence was observed in the S. epidermidis membranes but not in those of P. denitrificans. The different behaviour of both bacterial preparations is attributable to the more specific finding of ubiquinone in the particles of P. denitrificans.