myxothiazol and ubiquinol

Since available structures of native bc(1) complexes show a vacant Q(o)-site, occupancy by substrate and product must be investigated by kinetic and spectroscopic approaches. In this brief review, we discuss recent advances using these approaches that throw new light on the mechanism. The rate-limiting reaction is the first electron transfer after formation of the enzyme-substrate complex at the Q(o)-site. This is formed by binding of both ubiquinol (QH(2)) and the dissociated oxidized iron-sulfur protein (ISP(ox)). A binding constant of approximately 14 can be estimated from the displacement of E(m) or pK for quinone or ISP(ox), respectively. The binding likely involves a hydrogen bond, through which a proton-coupled electron transfer occurs. An enzyme-product complex is also formed at the Q(o)-site, in which ubiquinone (Q) hydrogen bonds with the reduced ISP (ISPH). The complex has been characterized in ESEEM experiments, which detect a histidine ligand, likely His-161 of ISP (in mitochondrial numbering), with a configuration similar to that in the complex of ISPH with stigmatellin. This special configuration is lost on binding of myxothiazol. Formation of the H-bond has been explored through the redox dependence of cytochrome c oxidation. We confirm previous reports of a decrease in E(m) of ISP on addition of myxothiazol, and show that this change can be detected kinetically. We suggest that the myxothiazol-induced change reflects loss of the interaction of ISPH with Q, and that the change in E(m) reflects a binding constant of approximately 4. We discuss previous data in the light of this new hypothesis, and suggest that the native structure might involve a less than optimal configuration that lowers the binding energy of complexes formed at the Q(o)-site so as to favor dissociation. We also discuss recent results from studies of the bypass reactions at the site, which lead to superoxide (SO) production under aerobic conditions, and provide additional information about intermediate states.

Topics: Benzoquinones; Binding Sites; Electron Transport Complex III; Iron-Sulfur Proteins; Kinetics; Methacrylates; Oxidation-Reduction; Thermodynamics; Thiazoles; Ubiquinone

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

Although several X-ray structures have been determined for the mitochondrial cytochrome (cyt) bc(1) complex, none yet shows the position of the substrate, ubiquinol, in the quinol oxidase (Q(o)) site. In this study, the interaction of molecular oxygen with the reactive intermediate Q(o) semiquinone is used to probe the Q(o) site. It has been known for some time that partial turnover of the cyt bc(1) complex in the presence of antimycin A, a Q(i) site inhibitor, results in accumulation of a semiquinone at the Q(o) site, which can reduce O(2) to superoxide (O(2)(*)(-)). It was more recently shown that myxothiazol, which binds close to the cyt b(L) heme in the proximal Q(o) niche, also induces O(2)(*)(-) production. In this work, it is shown that, in addition to myxothiazol, a number of other proximal Q(o) inhibitors [including (E)-beta-methoxyacrylate-stilbene, mucidin, and famoxadone] also induce O(2)(*)(-) production in the isolated yeast cyt bc(1) complex, at approximately 50% of the V(max) observed in the presence of antimycin A. It is proposed that proximal Q(o) site inhibitors induce O(2)(*)(-) production because they allow formation, but not oxidation, of the semiquinone at the distal niche of the Q(o) site pocket. The apparent K(m) for ubiquinol at the Q(o) site in the presence of proximal Q(o) site inhibitors suggests that the "distal niche" of the Q(o) pocket can act as a fully independent quinol binding and oxidation site. Together with the X-ray structures, these results suggest substrate ubiquinol binds in a fashion similar to that of stigmatellin with H-bonds between H161 of the Rieske iron-sulfur protein and E272 of the cyt b protein. When modeled in this way, mucidin and ubiquinol can bind simultaneously to the Q(o) site with virtually no steric hindrance, whereas progressively bulkier inhibitors exhibit increasing overlap. The fact that partial turnover of the Q(o) site is possible even with bound proximal Q(o) site inhibitors is consistent with the participation of two separate functional Q(o) binding niches, occupied simultaneously or sequentially.

Topics: Animals; Antimycin A; Benzoquinones; Binding Sites; Cattle; Crystallography, X-Ray; Electron Transport Complex III; Enzyme Inhibitors; Fungal Proteins; Heme; Hydrogen Bonding; Kinetics; Methacrylates; Models, Molecular; Oxygen; Protein Binding; Software; Superoxides; Thiazoles; Ubiquinone

The cytochrome (cyt) bc(1) complex is central to energy transduction in many species. Most investigators now accept a modified Q-cycle as the catalytic mechanism of this enzyme. Several thermodynamically favorable side reactions must be minimized for efficient functioning of the Q-cycle. Among these, reduction of oxygen by the Q(o) site semiquinone to produce superoxide is of special pathobiological interest. These superoxide-producing bypass reactions are most notably observed as the antimycin A- or myxothiazol-resistant reduction of cyt c. In this work, we demonstrate that these inhibitor-resistant cyt c reductase activities are largely unaffected by removal of O(2) in the isolated yeast cyt bc(1) complex. Further, increasing O(2) tension 5-fold stimulated the antimycin A-resistant reduction by a small amount ( approximately 25%), while leaving the myxothiazol-resistant reduction unchanged. This most likely indicates that the rate-limiting step in superoxide production is the formation of a reactive species (probably a semiquinone), capable of rapid O(2) reduction, and that in the absence of O(2) this species can reduce cyt c by some other pathway. We suggest as one possibility that a semiquinone escapes from the Q(o) site and reduces either O(2) or cyt c directly. The small increase in antimycin A-resistant cyt c reduction rate at high O(2) can be explained by the accumulation of a low concentration of a semiquinone inside the Q(o) site. Under aerobic conditions, addition of saturating levels of superoxide dismutase (SOD) inhibited 50% of cyt c reduction in the presence of myxothiazol, implying that essentially all bypass reactions occur with the production of superoxide. However, SOD inhibited only 35% of antimycin A-resistant cyt c reduction, suggesting the presence of a second, slower bypass reaction that does not reduce O(2). Given that myxothiazol blocks cyt b reduction whereas antimycin A promotes it, we propose that this second bypass occurs by reduction of the Q(o) site semiquinone by prereduced cyt b(L).

Topics: Aerobiosis; Anaerobiosis; Antimycin A; Cytochrome c Group; Electron Transport; Electron Transport Complex III; Enzyme Inhibitors; Methacrylates; Oxidation-Reduction; Oxidoreductases; Saccharomyces cerevisiae; Superoxides; Thiazoles; Ubiquinone

The role of the conserved acidic residues of subunit III of cytochrome c oxidase (COIII) in energy transduction was investigated. Using a COIII deletion mutant of Paracoccus denitrificans, complemented with a plasmid expressing either the wild type (wt) COIII gene or site-directed mutants of the COIII gene, we measured cytochrome c oxidase-dependent ATP synthesis, respiration, and membrane potential. Cytochrome c oxidase-dependent ATP synthesis was attenuated in nonacidic mutants of either Glu98 (E98A and E98Q), or Asp259 (D259A) but not in the acidic mutant E98D. The rates of respiration in the energy conversion-defective mutants were as high as or higher than that in the wt. The cytochrome c oxidase-induced increment of membrane potential in the nonacidic mutants was similar to or higher than that in the wt. In contrast, when succinate-driven ATP synthesis was mediated solely by ubiquinol oxidase (e.g., in the presence of myxothiazol), the rates of ATP synthesis in the nonacidic mutants were higher than that in the wt. Moreover, myxothiazol, which inhibited succinate respiration as well as ATP synthesis in wt and E98D, stimulated ATP synthesis, while inhibiting succinate respiration, in the nonacidic mutants. These results indicate that the attenuation of energy conversion in these mutants is limited to cytochrome c oxidase and thus suggest that subunit III plays a role in energy conversion by cytochrome c oxidase.

Topics: Adenosine Triphosphate; Ascorbic Acid; Electron Transport Complex IV; Membrane Potentials; Methacrylates; Mutation; Oxidative Phosphorylation; Oxygen Consumption; Paracoccus; Succinates; Succinic Acid; Tetramethylphenylenediamine; Thiazoles; Ubiquinone

Antimycin A and UHBDT inhibit the activity of the purified cytochrome bd complex from Azotobacter vinelandii. Inhibition of activity is non-competitive and antimycin A binding induces a shift to the red in the spectrum of a b-type haem. No inhibitory effects were seen with myxothiazol. Steady-state experiments indicate that the site of inhibition for antimycin A lies on the low-potential side of haem b558. In the presence of antimycin A at concentrations sufficient to inhibit respiration, some direct electron transfer from ubiquinol-1 to haem b595 and haem d still occurs. The results are consistent with a branched electron transfer pathway from ubiquinol to the oxygen reduction site.

Topics: Antimycin A; Azotobacter vinelandii; Cytochrome b Group; Cytochromes; Dithiothreitol; Electron Transport; Electron Transport Chain Complex Proteins; Escherichia coli Proteins; Kinetics; Methacrylates; Oxidation-Reduction; Oxidoreductases; Thiazoles; Ubiquinone

Topics: Antifungal Agents; Binding Sites; Electron Transport Complex III; Kinetics; Methacrylates; Mutagenesis, Site-Directed; Oxidation-Reduction; Point Mutation; Recombinant Proteins; Saccharomyces cerevisiae; Thiazoles; Ubiquinone

The cytochrome bc1 complexes from the nonphotosynthetic strain R126 of Rhodobacter capsulatus and from its revertant MR126 were purified. Between both preparations, no difference could be observed in the stoichiometries of the cytochromes, in their spectral properties, and in their midpoint redox potentials. Both also showed identical polypeptide patterns after electrophoresis on polyacrylamide gels in the presence of sodium dodecylsulfate. The ubiquinol: cytochrome c oxidoreductase activity was strongly inhibited in the complex from the mutant compared to the one from the revertant. So was the oxidant-induced extra reduction of cytochrome b. Both preparations, however, showed an antimycin-induced red shift of cytochrome b, as well as antimycin-sensitive reduction of cytochrome b by ubiquinol. In accordance with a preceding study of chromatophores (Robertson et al. (1986). J. Biol. Chem. 261, 584-591), it is concluded that the mutation affects specifically the ubiquinol oxidizing site, leaving the ubiquinol reducing site unchanged.

Topics: Antimycin A; Catalysis; Centrifugation, Density Gradient; Electron Transport Complex III; Electrophoresis, Polyacrylamide Gel; Immunoblotting; Methacrylates; Mutation; Oxidation-Reduction; Rhodobacter capsulatus; Spectrum Analysis; Thermodynamics; Thiazoles; Ubiquinone

The interaction of the photosynthetic reaction center (RC)-generated ubiquinol with the ubiquinone-reducing center C of ubiquinol:cytochrome c2-oxidoreductase (bc1-complex) has been studied electrometrically in Rhodobacter sphaeroides chromatophores. The addition of myxothiazol inhibited the ubiquinol-oxidizing center Z, suppressing the phases of membrane potential generation by the bc1-complex, but at the same time induced an electrogenic phase of opposite polarity, sensitive to antimycin A, the inhibitor of center C. The rise time of this reverse phase varied from 3 ms at pH 6.0 to 1 ms at pH 9.5. At pH greater than 9.5 the reverse phase was limited by the rate of ubiquinol formation in RC. The magnitude of the reverse phase was constant within the pH range 7.5-10.0. It is assumed that the reverse phase is due to the electrogenic deprotonation reaction which takes place after the binding of the RC-generated ubiquinol to center C.

Topics: Cytochrome c Group; Cytochromes c2; Electron Transport Complex III; Hydrogen-Ion Concentration; Membrane Potentials; Methacrylates; Photosynthesis; Photosynthetic Reaction Center Complex Proteins; Rhodobacter sphaeroides; Thiazoles; Ubiquinone

Seven single-site mutants in six residues of the cyt b polypeptide of Rhodobacter capsulatus selected for resistance to the Qo site inhibitors stigmatellin, myxothiazol, or mucidin [Daldal, F., Tokito, M.K., Davidson, E., & Faham, M. (1989) EMBO J. 8, 3951-3961] have been characterized by using optical and EPR spectroscopy and single-turnover kinetic analysis. The strains were compared with wild-type strain MT1131 and with the Ps- strain R126 (G158D), which is dysfunctional in its Qo site [Robertson, D.E., Davidson, E., Prince, R.C., van den Berg, W.H., Marrs, B.L., & Dutton, P.L. (1986) J. Biol. Chem. 261, 584-591]. Mutants selected for stigmatellin resistance induced a weakening in the binding of the inhibitor without discernible loss of ubiquinone(Q)/ubiquinol(QH2) binding affinity to the Qo site or kinetic impairment to catalysis. Mutants selected for myxothiazol or mucidin resistance, inducing weakening of inhibitor binding, all displayed impaired rates of Qo site catalysis: The most severe cases (F144L, F144S) displayed loss of affinity for Q, and evidence suggests that parallel loss of affinity for the substrate QH2 was incurred in these strains. The results provide a view of the nature of the interaction of Q and QH2 of the Qpool with the Qo site. Consideration of the mutational substitutions and their structural positions along with comparisons with the QA and QB sites of the photosynthetic reaction center suggests a model for the structure of the Qo site.

Topics: Amino Acid Sequence; Anti-Bacterial Agents; Cytochrome b Group; Electron Spin Resonance Spectroscopy; Electron Transport; Electron Transport Complex III; Fatty Acids, Unsaturated; Kinetics; Methacrylates; Models, Structural; Molecular Sequence Data; Mutation; Oxidation-Reduction; Polyenes; Protein Conformation; Rhodobacter capsulatus; Strobilurins; Thiazoles; Ubiquinone

A non-photosynthetic mutant (Ps-) of Rhodopseudomonas capsulata, designated R126, was analyzed for a defect in the cyclic electron transfer system. Compared to a Ps+ strain MR126, the mutant was shown to have a full complement of electron transfer components (reaction centers, ubiquinone-10, cytochromes b, c1, and c2, the Rieske 2-iron, 2-sulfur (Rieske FeS) center, and the antimycin-sensitive semiquinone). Functionally, mutant R126 failed to catalyze complete cytochrome c1 + c2 re-reduction or cytochrome b reduction following a short (10 microseconds) flash of actinic light. Evidence (from flash-induced carotenoid band shift) was characteristic of inhibition of electron transfer proximal to cytochrome c1 of the ubiquinol-cytochrome c2 oxidoreductase. Three lines of evidence indicate that the lesion of R126 disrupts electron transfer from quinol to Rieske FeS: 1) the degree of cytochrome c1 + c2 re-reduction following a flash is indicative of electron transfer from Rieske FeS to cytochrome c1 + c2 without redox equilibration with an additional electron from a quinol; 2) inhibitors that act at the Qz site and raise the Rieske FeS midpoint redox potential (Em), namely 5-undecyl-6-hydroxy-4,7-dioxobenzothiazole or 3-alkyl-2-hydroxy-1,4-napthoquinone, have no effect on cytochrome c1 + c2 oxidation in R126; 3) the Rieske FeS center, although it exhibits normal redox behavior, is unable to report the redox state of the quinone pool, as metered by its EPR line shape properties. Flash-induced proton binding in R126 is indicative of normal functional primary (QA) and secondary (QB) electron acceptor activity of the photosynthetic reaction center. The Qc functional site of cytochrome bc1 is intact in R126 as measured by the existence of antimycin-sensitive, flash-induced cytochrome b reduction.

Topics: Antimycin A; Benzoquinones; Cytochrome c Group; Electron Spin Resonance Spectroscopy; Electron Transport; Electron Transport Complex III; Methacrylates; Multienzyme Complexes; Mutation; Oxidation-Reduction; Photolysis; Quinone Reductases; Quinones; Rhodopseudomonas; Thiazoles; Ubiquinone

Stigmatellin, a chromone inhibitor acting at the Q0 center of the bc1 complex, binds to the heme b-566 domain of cytochrome b as well as to the Fe2S2 protein. Its binding induces a shift of the alpha-band of heme b-566 to 568 nm. It does not influence the ligand field of the heme b-562 center. Concomitant with the red shift, stigmatellin gives rise to an alteration of the EPR line shape of the Fe2S2 cluster, namely linewidth narrowing and g value shifts at all 3 principal values. The midpoint redox potential of the Fe2S2 protein is shifted from 290 to 540 mV.

Topics: Animals; Binding Sites; Binding, Competitive; Cattle; Electron Spin Resonance Spectroscopy; Electron Transport Complex III; Intracellular Membranes; Iron-Sulfur Proteins; Methacrylates; Mitochondria; Multienzyme Complexes; Oxidation-Reduction; Polyenes; Protein Binding; Quinone Reductases; Thiazoles; Ubiquinone

Myxothiazol, an antibiotic from Myxococcus fulvus, which inhibits mitochondrial respiration in the bc1 complex of the respiratory chain, has effects on the redox components of isolated succinate-cytochrome c reductase complex which suggest that it interacts with both cytochrome b and the iron-sulfur protein of the bc1 complex. The inhibitor appears to increase the midpoint potentials of cytochromes b-562 and b-566, as indicated by an increase in their reducibility by the succinate/fumarate couple. It also causes a red shift in the optical spectrum of ferrocytochrome b-566, as reported previously (Becker, W. F., Von Jagow , G., Anke , T., Steglisch , W. (1981) FEBS Lett. 132, 329-333). This red shift is enhanced by Triton X-100, and there is no shift in the spectrum of b-562. These results are consistent with evidence that mutations conferring myxothiazol resistance in yeast map to the mitochondrial gene for cytochrome b ( Thierbach , G., and Michaelis, G. (1982) Mol. Gen. Genet. 186, 501-506). In addition, myxothiazol has effects on reduction of the cytochromes b and c1 by succinate or ubiquinol which are identical to those caused by removal of the iron-sulfur protein from the bc1 complex. It blocks reduction of cytochrome c1 during single and multiple turnovers of the bc1 complex, but does not block reduction of the b cytochromes. In the presence of antimycin, it blocks reduction of both cytochromes b and c1. In contrast to antimycin, myxothiazol inhibits oxidant-induced reduction of both b cytochromes and does not inhibit their oxidation by fumarate. Myxothiazol also inhibits reduction of the iron-sulfur protein by ubiquinol and shifts the gx resonance in the EPR spectrum of the iron-sulfur protein from g = 1.79 to 1.76. It does not affect the midpoint potential of the iron-sulfur protein, but does eliminate the increase in midpoint potential which is caused by inhibitory hydroxyquinones which bind to the iron-sulfur protein. The effects of myxothiazol are consistent with a protonmotive Q cycle pathway of electron transfer in which myxothiazol binds to cytochrome b and displaces quinone from the iron-sulfur protein of the bc1 complex. These results suggest either that a myxothiazol-induced conformational change in cytochrome b is transmitted to a quinone binding site on the iron-sulfur protein, or that there is a quinone binding site which consists of peptide domains from both cytochrome b and iron-sulfur protein.

Topics: Animals; Antifungal Agents; Cattle; Cytochrome b Group; Cytochromes c1; Electron Transport Complex III; Iron-Sulfur Proteins; Kinetics; Metalloproteins; Methacrylates; Mitochondria, Heart; Multienzyme Complexes; NADH, NADPH Oxidoreductases; Oxygen Consumption; Quinone Reductases; Quinones; Succinates; Succinic Acid; Thiazoles; Ubiquinone

Topics: Animals; Antifungal Agents; Antimycin A; Binding Sites; Cattle; Cytochrome b Group; Cytochromes; Electron Transport; Kinetics; Methacrylates; Oxidation-Reduction; Spectrophotometry, Ultraviolet; Spectrum Analysis; Submitochondrial Particles; Thiazoles; Ubiquinone