chiniofon and myxothiazol

It was found that Acidithiobacillus thiooxidans has sulfite:ubiquinone oxidoreductase and ubiquinol oxidase activities in the cells. Ubiquinol oxidase was purified from plasma membranes of strain NB1-3 in a nearly homogeneous state. A purified enzyme showed absorption peaks at 419 and 595 nm in the oxidized form and at 442 and 605 nm in the reduced form. Pyridine ferrohaemochrome prepared from the enzyme showed an alpha-peak characteristic of haem a at 587 nm, indicating that the enzyme contains haem a as a component. The CO difference spectrum of ubiquinol oxidase showed two peaks at 428 nm and 595 nm, and a trough at 446 nm, suggesting the existence of an aa(3)-type cytochrome in the enzyme. Ubiquinol oxidase was composed of three subunits with apparent molecular masses of 57 kDa, 34 kDa, and 23 kDa. The optimum pH and temperature for ubiquinol oxidation were pH 6.0 and 30 degrees C. The activity was completely inhibited by sodium cyanide at 1.0 mM. In contrast, the activity was inhibited weakly by antimycin A(1) and myxothiazol, which are inhibitors of mitochondrial bc(1) complex. Quinone analog 2-heptyl-4-hydoroxyquinoline N-oxide (HOQNO) strongly inhibited ubiquinol oxidase activity. Nickel and tungstate (0.1 mM), which are used as a bacteriostatic agent for A. thiooxidans-dependent concrete corrosion, inhibited ubiquinol oxidase activity 100 and 70% respectively.

Topics: Acidithiobacillus thiooxidans; Antimycin A; Cell Membrane; Electron Transport Complex IV; Heme; Hydrogen-Ion Concentration; Hydroxyquinolines; Methacrylates; Nickel; Oxidation-Reduction; Oxidoreductases; Protein Subunits; Sodium Cyanide; Sulfites; Thiazoles; Tungsten Compounds; Ubiquinone

We tested three oxidant sensitive fluoroprobes (dihydrorhodamine [DHR], 2',7'-dichlorodihydrofluorescein [H(2)DCF], and dihydroethidium [DHE]) for interactions with three inhibitors of mitochondrial electron transport. DHR, H(2)DCF, and DHE produced large time-dependent increases in fluorescence in a cell-free medium that contained either of the site III inhibitors antimycin (A) and 2-heptyl-4-hydroxy-quinoline-N-oxide but minimal increases in medium that contained another site III inhibitor, myxothiazol (Mx). The interactions between A and each of the fluoroprobes occurred at concentrations of agent/probe that are frequently used in experiments designed to investigate cellular oxidant production. To define more effectively the nature of these agent/probe interactions, we determined the oxygen dependence of the interactions between A and each probe. The A/H(2)DCF and A/DHR interactions either were highly oxygen-dependent or exhibited a small degree of oxygen dependence, respectively, whereas the A/DHE interaction was oxygen-independent. Finally, we determined multiple ways to reduce the impact of the agent/probe interaction on data acquisition. The addition of either fetal bovine serum (10%) or albumin (5%) to the media abolished the A/DHR and A/H(2)DCF interactions. Shifting the excitation wavelength of DHE (from 470 to 530 nm) reduced measurement of the A/DHE interaction while preserving measurement of the intracellular signal. Collectively, these results emphasize the importance of testing for interactions between agents and probes, because these interactions can interfere with the accurate interpretation of experimental results. In addition, the methods presented for circumventing these interactions may be applicable to other experiments in which agent/probe interactions are an obstacle to accurate interpretation of the experimental results.

Topics: Antimycin A; Cells, Cultured; Electron Transport; Endothelium, Vascular; Fluorescent Dyes; Humans; Hydroxyquinolines; Indicators and Reagents; Methacrylates; Oxidants; Thiazoles; Time Factors

The redox components of the cytochrome bc1 complex from the acidophilic chemolithotrophic organism Thiobacillus ferrooxidans were investigated by potentiometric and spectroscopic techniques. Optical redox titrations demonstrated the presence of two b-type hemes with differing redox midpoint potentials at pH 7.4 (-169 and + 20 mV for bL and bH, respectively). At pH 3.5, by contrast, both hemes appeared to titrate at about +20 mV. Antimycin A, 2-heptyl-4-hydroxyquinoline N-oxide, and stigmatellin induced distinguishable shifts of the b hemes' alpha-bands, providing evidence for the binding of antimycin A and 2-heptyl-4-hydroxyquinoline N-oxide near heme bH (located on the cytosolic side of the membrane) and of stigmatellin near heme bL (located on the periplasmic side of the membrane). The inhibitors stigmatellin, 5-(n-undecyl)-6-hydroxy-4,7-dioxobenzothiazole, and 2, 5-dibromo-3-methyl-6-isopropyl-p-benzoquinone affected the EPR spectrum of the Rieske iron-sulfur center in a way that differs from what has been observed for cytochrome bc1 or b6f complexes. The results obtained demonstrate that the T. ferrooxidans complex, although showing most of the features characteristic for bc1 complexes, contains unique properties that are most probably related to the chemolithotrophicity and/or acidophilicity of its parent organism. A speculative model for reverse electron transfer through the T. ferrooxidans complex is proposed.

Topics: Antimycin A; Electron Spin Resonance Spectroscopy; Electron Transport Complex III; Ferrous Compounds; Heme; Hydrogen-Ion Concentration; Hydroxyquinolines; Iron; Iron-Sulfur Proteins; Methacrylates; Models, Chemical; Oxidation-Reduction; Polyenes; Potentiometry; Proton-Motive Force; Species Specificity; Spectrophotometry; Thermodynamics; Thiazoles; Thiobacillus

1. Electron transport components involved in nitrous oxide reduction in several strains of Rhodobacter capsulatus and in the denitrifying strain of Rhodobacter sphaeroides (f. sp. denitrificans) have been investigated. Detailed titrations with antimycin A and myxothiazol, inhibitors of the cytochrome bc1 complex, show that part of the electron flow to nitrous oxide passes through this complex. The sensitivity to myxothiazol varies between strains and growth conditions of R. capsulatus; the higher rates of nitrous oxide reduction correlate with the higher sensitivities. Partial inhibition of the nitrous oxide reductase enzyme with azide decreased the sensitivity to myxothiazol of the strains that had the highest nitrous oxide reductase activity. 2. Inhibition of nitrous oxide reduction in cells of R. capsulatus by myxothiazol could be restored under dark conditions by addition of N,N,N',N'-tetramethyl-p-phenylene diamine. The highest activities observed after addition of this electron carrier were found in the strains that had the highest sensitivity to myxothiazol, consistent with the premise that this inhibitor is more effective at the higher flux rates to nitrous oxide. 3. Addition of nitrous oxide to cells of R. capsulatus strain N22DNAR+ under darkness caused oxidation of both b- and c-type cytochromes. The oxidation of b cytochromes was less pronounced in the presence of myxothiazol, consistent with a role for the cytochrome bc1 complex in the electron pathway to nitrous oxide. Ferricyanide, in the absence of myxothiazol, caused a similar extent of oxidation of b cytochromes, but a greater oxidation of c-type, suggesting that there was a pool of c-type cytochrome that was not oxidisable by nitrous oxide. The time course showed that both the b- and c-type cytochromes were oxidised within a few seconds of the addition of nitrous oxide. During the following seconds there was a partial re-reduction of the cytochromes such that after approximately 1 min a lower steady-state of oxidation was attained and this persisted until the nitrous oxide was exhausted. 4. A mutant, MTCBC1, of R. capsulatus that specifically lacked a functional cytochrome bc1 complex reduced nitrous oxide, albeit at 30% of the rate shown by the parent strain MT1131. A reduced minus nitrous-oxide-oxidised difference spectrum for MTCBC1 in the absence of myxothiazol was similar to the corresponding difference spectrum observed for strain N22DNAR+ in the presence of myxothiazol. It is sug

Topics: Anaerobiosis; Antimycin A; Darkness; Electron Transport; Electron Transport Complex III; Hydroxyquinolines; Membrane Potentials; Methacrylates; Nitrous Oxide; Oxidation-Reduction; Rhodospirillaceae; Thiazoles

Changes are described that are brought about by antimycin, NoHOQnO, funiculosin, myxothiazol and mucidin in the alpha-, beta- and gamma-absorption bands of reduced and oxidized cytochromes b in the isolated complex bc1 form beef heart mitochondria. The inhibitors can be divided into 2 groups. Antimycin, funiculosin and NoHOQnO are likely to shift the spectrum of b-562 and compete for specific binding with complex bc1, with each other but not with myxothiazol and mucidin. The spectral effects of the latter two inhibitors are more difficult to interpret and may involve contributions not only from b-562 but from b-566 as well. The existence of 2 independent inhibitor binding-sites in the complex bc1 corroborates the Q-cycle hypothesis.

Topics: Alkenes; Animals; Anthraquinones; Antimycin A; Cattle; Cytochrome b Group; Electron Transport; Electron Transport Complex III; Fatty Acids, Unsaturated; Hydroxyquinolines; Methacrylates; Mitochondria, Heart; Multienzyme Complexes; NADH, NADPH Oxidoreductases; Quinone Reductases; Spectrophotometry; Strobilurins; Thiazoles

Bovine-heart Complex III can catalyze the reduction of spinach plastocyanin by a decyl analog of ubiquinol-2 at a rate comparable with the rate of plastocyanin reduction by plastoquinol as catalyzed by the cytochrome b6-f complex purified from spinach leaves. This plastocyanin reduction as catalyzed by Complex III was almost completely inhibited by myxothiazol at stoichiometric concentrations, partially inhibited by UHDBT (5-n-undecyl-6-hydroxy-4,7-dioxobenzothiazole) and funiculosin, and was relatively insensitive to antimycin and HQNO (2-n-heptyl-4-hydroxyquinoline-N-oxide). Cytochrome c reduction as catalyzed by Complex III displayed a residual, inhibitor-insensitive rate of 5% of the uninhibited rate for each of the three inhibitors, antimycin, myxothiazol, and UHDBT. However, the residual rate that was insensitive to each of the inhibitors added singly was inhibited further by addition of the remaining two inhibitors. From these results it is concluded that plastocyanin reduction involves an electron-transfer pathway through Complex III that is distinct from the pathway utilized for reduction of cytochrome c.

Topics: Animals; Anthraquinones; Antimycin A; Cattle; Drug Resistance; Electron Transport; Electron Transport Complex III; Hydrogen-Ion Concentration; Hydroxyquinolines; Methacrylates; Models, Chemical; Multienzyme Complexes; Myocardium; NADH, NADPH Oxidoreductases; Oxidation-Reduction; Plant Proteins; Plastocyanin; Quinone Reductases; Thiazoles

Antimycin, 2-nonyl-4-hydroxyquinoline N-oxide and funiculosin induce O.2(-) generation by submitochondrial particles oxidizing succinate, whereas KCN, mucidin, myxothiazol or 2,3-dimercaptopropanol inhibit O.2(-) generation. Thenoyltrifluoroacetone does not induce superoxide production by itself but slightly stimulates the reaction initiated by antimycin. The results indicate that auto-oxidation of unstable ubisemiquinone formed in centre o of the Q-cycle generates most of the O.2(-) radicals in the cytochrome bc1-site of the mitochondrial respiratory chain.

Topics: Alkenes; Animals; Anthraquinones; Antimycin A; Cattle; Dimercaprol; Electron Transport Complex III; Fatty Acids, Unsaturated; Hydroxyquinolines; Methacrylates; Mitochondria, Heart; Multienzyme Complexes; NADH, NADPH Oxidoreductases; Oxygen; Quinone Reductases; Strobilurins; Superoxides; Thenoyltrifluoroacetone; Thiazoles

Anaerobic potentiometric titrations of b cytochromes have been carried out in beef heart submitochondrial particles in the presence of several specific inhibitors of electron transfer through the b-c1-site of the respiratory chain. Whereas antimycin shows no significant effect on the titration curve of cytochrome b-562, NoHOQnO is found to shift the Em of b-562 by 20-30 mV to the positive. Funiculosin raises the Em of b-562 by greater than 100 mV and also appears to bring about a minor shift of b-566 midpoint potential. In the presence of myxothiazol, both b cytochromes titrate with Em values 15-30 mV more positive than in the control.

Topics: Animals; Anthraquinones; Antimycin A; Binding Sites; Cattle; Cytochromes; Electron Transport Complex III; Hydroxyquinolines; Methacrylates; Mitochondria, Heart; Multienzyme Complexes; NADH, NADPH Oxidoreductases; Oxidation-Reduction; Potentiometry; Quinone Reductases; Thiazoles

Mouse LA9 cell lines were selected for increased resistance to either HQNO or myxothiazol, inhibitors of electron transport which bind to the mitochondrial cytochrome b protein. Two phenotypically distinguishable HQNO-resistant mutants were recovered while the myxothiazol-resistant isolates had a common phenotype. All three mutant phenotypes were transmitted cytoplasmically in cybrid crosses. Biochemical studies further established that for all three mutant types, resistance at the cellular level was paralleled by an increase in inhibitor resistance of mitochondrial succinate-cytochrome c oxidoreductase, the respiratory complex containing cytochrome b. As with the previously described mitochondrial antimycin-resistant mutant, the initial biochemical and genetic studies indicated that these mutations occur within the mitochondrial cytochrome b gene. This conclusion was strongly supported by the results of mtDNA restriction fragment analyses in which it was found that one HQNO-resistant mutant had undergone a small insertion or duplication in the apocytochrome b gene. Finally, all four mitochondrial cytochrome b mutants have been analyzed in both cell plating studies and succinate-cytochrome c oxidoreductase assays to determine the pattern of cross-resistance to inhibitors of cytochrome b other than the one used for selection.

Topics: Animals; Cell Line; DNA, Mitochondrial; Drug Resistance; Gene Expression Regulation; Genes; Hydroxyquinolines; Methacrylates; Mice; Mitochondria; Mutation; Oxidoreductases; Phenotype; Succinate Cytochrome c Oxidoreductase; Thiazoles