ubiquinone and myxothiazol

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

Yeast Ndi1 is a monotopic alternative NADH dehydrogenase. Its crystal structure in complex with the electron acceptor, ubiquinone, has been determined. However, there has been controversy regarding the ubiquinone binding site. To address these points, we identified the first competitive inhibitor of Ndi1, stigmatellin, along with new mixed-type inhibitors, AC0-12 and myxothiazol, and thereby determined the crystal structures of Ndi1 in complexes with the inhibitors. Two separate binding sites of stigmatellin, STG-1 and STG-2, were observed. The electron density at STG-1, located at the vicinity of the FAD cofactor, further demonstrated two binding modes: STG-1a and STG-1b. AC0-12 and myxothiazol are also located at the vicinity of FAD. The comparison of the binding modes among stigmatellin at STG-1, AC0-12, and myxothiazol revealed a unique position for the aliphatic tail of stigmatellin at STG-1a. Mutations of amino acid residues that interact with this aliphatic tail at STG-1a reduced the affinity of Ndi1 for ubiquinone. In conclusion, the position of the aliphatic tail of stigmatellin at STG-1a provides a structural basis for its competitive inhibition of Ndi1. The inherent binding site of ubiquinone is suggested to overlap with STG-1a that is distinct from the binding site for NADH.

Topics: Amino Acid Sequence; Binding Sites; Binding, Competitive; Coenzymes; Crystallography, X-Ray; Electron Transport Complex I; Flavin-Adenine Dinucleotide; Gene Expression; Kinetics; Methacrylates; Models, Molecular; Mutation; Polyenes; Protein Binding; Protein Conformation, alpha-Helical; Protein Conformation, beta-Strand; Protein Interaction Domains and Motifs; Recombinant Proteins; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Sequence Alignment; Sequence Homology, Amino Acid; Substrate Specificity; Thermodynamics; Thiazoles; Ubiquinone

Bexsero, a new vaccine against Neisseria meningitidis serogroup B (MenB), is composed of 3 main recombinant proteins and an outer membrane vesicle component. One of the main bactericidal antigens, neisseria heparin binding antigen (NHBA), is present as a fusion protein with the accessory protein genome-derived neisserial antigen (GNA) 1030 to further increase its immunogenicity. The gene encoding for GNA1030 is present and highly conserved in all Neisseria strains, and although orthologs are present in numerous species, its biologic function is unknown. Native mass spectrometry was used to demonstrate that GNA1030 forms a homodimer associated with 2 molecules of ubiquinone-8 (Ub8), a cofactor mainly involved in the electron transport chain and with antioxidant properties. Disc diffusion assays on the wild-type and knockout mutant of GNA1030, in the presence of various compounds, suggested that GNA1030 is not involved in oxidative stress or electron chain transport per se, although it contributes to constitutive refilling of the inner membrane with Ub8. These studies shed light on an accessory protein present in Bexsero and reveal functional insights into the family of related proteins. On the basis of our findings, we propose to name the protein neisseria ubiquinone binding protein (NUbp).

Topics: Amino Acid Sequence; Anti-Bacterial Agents; Antigens, Bacterial; Antimycin A; Bacterial Proteins; Blotting, Western; Cell Survival; Cloning, Molecular; Disulfides; Electron Transport Complex III; Hydrogen Peroxide; Mass Spectrometry; Meningococcal Vaccines; Methacrylates; Molecular Sequence Data; Mutation; Neisseria meningitidis; Oxidants; Periplasmic Proteins; Protein Binding; Protein Multimerization; Thiazoles; Ubiquinone

The effects of decylubiquinone, a ubiquinone analogue, on mitochondrial function and inhibition thresholds of the electron transport chain enzyme complexes in synaptosomes were investigated. Decylubiquinone increased complex I/III and complex II/III activities by 64 and 80%, respectively, and attenuated reductions in oxygen consumption at high concentrations of the complex III inhibitor myxothiazol. During inhibition of complex I, decylubiquinone attenuated reductions in synaptosomal oxygen respiration rates, as seen in the complex I inhibition threshold. Decylubiquinone increased the inhibition thresholds of complex I/III, complex II/III, and complex III over oxygen consumption in the nerve terminal by 25-50%, when myxothiazol was used to inhibit complex III. These results imply that decylubiquinone increases mitochondrial function in the nerve terminal during complex I or III inhibition. The potential benefits of decylubiquinone in diseases where complex I, I/III, II/III, or III activities are deficient are discussed.

Topics: Animals; Antimycin A; Electron Transport; Female; Methacrylates; Mitochondria; Models, Biological; Neurodegenerative Diseases; Oxygen Consumption; Rats; Rats, Wistar; Rotenone; Synaptosomes; Thiazoles; Ubiquinone; Uncoupling Agents

Deletion of COQ10 in Saccharomyces cerevisiae elicits a respiratory defect characterized by the absence of cytochrome c reduction, which is correctable by the addition of exogenous diffusible coenzyme Q(2). Unlike other coq mutants with hampered coenzyme Q(6) (Q(6) ) synthesis, coq10 mutants have near wild-type concentrations of Q(6). In the present study, we used Q-cycle inhibitors of the coenzyme QH(2)-cytochrome c reductase complex to assess the electron transfer properties of coq10 cells. Our results show that coq10 mutants respond to antimycin A, indicating an active Q-cycle in these mutants, even though they are unable to transport electrons through cytochrome c and are not responsive to myxothiazol. EPR spectroscopic analysis also suggests that wild-type and coq10 mitochondria accumulate similar amounts of Q(6) semiquinone, despite a lower steady-state level of coenzyme QH(2)-cytochrome c reductase complex in the coq10 cells. Confirming the reduced respiratory chain state in coq10 cells, we found that the expression of the Aspergillus fumigatus alternative oxidase in these cells leads to a decrease in antimycin-dependent H(2)O(2) release and improves their respiratory growth.

Topics: Antifungal Agents; Antimycin A; Aspergillus fumigatus; Cytochrome c Group; Electron Spin Resonance Spectroscopy; Electron Transport; Hydrogen Peroxide; Immunoblotting; Methacrylates; Mitochondria; Mitochondrial Proteins; Mutation; Oxidation-Reduction; Oxidoreductases; Oxygen Consumption; Plant Proteins; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Superoxides; Thiazoles; Ubiquinone

The effect of hypericin photoactivation on mitochondria of human prostate carcinoma cells was studied using a range of mitochondrial inhibitors. Oligomycin significantly enhanced hypericin phototoxicity while atractyloside and antymicin A conferred a significant protection. Use of myxothiazol did not affect cell survival following hypericin photoactivation. These results signify a protective role for F(1)F(0)-ATP synthase running in reverse mode, and a significant photodamage at the quinone-reducing site of mitochondrial complex III. In light of these results, we performed molecular modeling of hypericin binding to complex III. This revealed three binding sites, two of which coincided with the quinol-oxidizing and quinone-reducing centers. Using submitochondrial particles we examined hypericin as a possible substrate of complex III and compared this to its natural substrate, ubiquinone-10. Our results demonstrate uniquely that hypericin is an efficient substrate for complex III, and this activity is inhibited by myxothiazol and antimycin A. We further demonstrated that hypericin photosensitization completely inactivated complex III with ubiquinone as substrate. The ability to enhance HYP potency by inhibition of F(1)F(0)-ATP synthase or depress HYP efficacy by inhibition at the Qi site of complex III provides a potential to increase the therapeutic index of HYP and amplify its PDT action in tumor cells.

Topics: Anthracenes; Antimycin A; Binding Sites; Cell Line, Tumor; Cytochromes c; Electron Transport Complex III; Humans; Light; Methacrylates; Mitochondria; Mitochondrial Proton-Translocating ATPases; Models, Molecular; Perylene; Spectrophotometry, Ultraviolet; Submitochondrial Particles; Thiazoles; Ubiquinone

Activity defects in respiratory chain complexes are responsible for a large variety of pathological situations, including neuromuscular diseases and multisystemic disorders. Their impact on energy production is highly variable and disproportional. The same biochemical or genetic defect can lead to large differences in clinical symptoms and severity between tissues and patients, making the pathophysiological analysis of mitochondrial diseases difficult. The existence of compensatory mechanisms operating at the level of the respiratory chain might be an explanation for the biochemical complexity observed for respiratory defects. Here, we analyzed the role of cytochrome c and coenzyme Q in the attenuation of complex III and complex IV pharmacological inhibition on the respiratory flux. Spectrophotometry, HPLC-EC, polarography and enzymology permitted the calculation of molar ratios between respiratory chain components, giving values of 0.8:61:3:12:6.8 in muscle and 1:131:3:9:6.5 in liver, for CII:CoQ:CIII:Cyt c:CIV. The results demonstrate the dynamic functional compartmentalization of respiratory chain substrates, with the existence of a substrate pool that can be recruited to maintain energy production at normal levels when respiratory chain complexes are inhibited. The size of this reserve was different between muscle and liver, and in proportion to the magnitude of attenuation of each respiratory defect. Such functional compartmentalization could result from the recently observed physical compartmentalization of respiratory chain substrates. The dynamic nature of the mitochondrial network may modulate this compartmentalization and could play a new role in the control of mitochondrial respiration as well as apoptosis.

Topics: Animals; Cytochromes c; Electron Transport; Electron Transport Complex III; Electron Transport Complex IV; Male; Methacrylates; Mitochondria, Liver; Mitochondria, Muscle; Mitochondrial Diseases; Oxygen Consumption; Potassium Cyanide; Rats; Rats, Wistar; 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

Reactive oxygen species (ROS) mediate volatile anesthetic preconditioning. We tested the hypothesis that isoflurane (ISO) generates ROS from electron transport chain complexes I and III. Rabbits (n = 55) underwent 30 min coronary artery occlusion followed by 3 h reperfusion and received 0.9% saline, the complex I inhibitor diphenyleneiodonium (DPI; 1.5 mg/kg bolus followed by 1.5 mg/kg over 1 h), or the complex III inhibitor myxothiazol (MYX; 0.1 mg/kg bolus followed by 0.3 mg/kg over 1 h) in the absence and presence of 1.0 minimum alveolar concentration ISO. ISO was administered for 30 min and discontinued 15 min before coronary occlusion. Infarct size and ROS production (n = 32) were determined using triphenyltetrazolium staining and ethidium-DNA fluorescence, respectively. Adenosine triphosphate (ATP) synthesis in mitochondria obtained from rabbit hearts (n = 24) subjected to drug interventions was measured by luciferin-luciferase luminometry. ISO significantly (P < 0.05) reduced infarct size (19% +/- 4%) as compared with control (39% +/- 4%). MYX (35% +/- 4%), but not DPI (24% +/- 2%), abolished this protection. ISO increased ethidium-DNA fluorescence (83 +/- 11 U) as compared with control (40 +/- 12 U). MYX (35 +/- 3 U), but not DPI (78 +/- 9 U), abolished ROS generation. DPI and MYX selectively reduced complex I- and complex III-mediated ATP synthesis, respectively. ROS generated from electron transport chain complex III mediate ISO-induced cardioprotection.

Topics: Adenosine Triphosphate; Anesthetics, Inhalation; Animals; Coenzymes; Electron Transport; Enzyme Inhibitors; Hemodynamics; In Vitro Techniques; Ischemic Preconditioning, Myocardial; Isoflurane; Male; Methacrylates; Mitochondria, Heart; Myocardial Infarction; NADH Dehydrogenase; Onium Compounds; Rabbits; Reactive Oxygen Species; Thiazoles; Ubiquinone; Ventricular Function, Left

Complex III in the mitochondrial electron transport chain is a proposed site for the enhanced production of reactive oxygen species that contribute to aging in the heart. We describe a defect in the ubiquinol binding site (Q(O)) within cytochrome b in complex III only in the interfibrillar population of cardiac mitochondria during aging. The defect is manifested as a leak of electrons through myxothiazol blockade to reduce cytochrome b and is observed whether cytochrome b in complex III is reduced from the forward or the reverse direction. The aging defect increases the production of reactive oxygen species from the Q(O) site of complex III in interfibrillar mitochondria. A greater leak of electrons from complex III during the oxidation of ubiquinol is a likely mechanism for the enhanced oxidant production from mitochondria that contributes to aging in the rat heart.

Topics: Aging; Animals; Antimycin A; Binding Sites; Cytochrome b Group; Electron Transport; Electron Transport Complex III; Enzyme Activation; Hydroquinones; In Vitro Techniques; Male; Methacrylates; Mitochondria, Heart; Mitochondrial Diseases; Myofibrils; Oxidation-Reduction; Polyenes; Rats; Reactive Oxygen Species; Thiazoles; 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 rotenone-insensitive NADH dehydrogenase isolated from mitochondria of the procyclic form of Trypanosoma brucei has the ability to produce superoxide anions (Biochemistry 41 (2002) 3065). Superoxide production by the purified enzyme was 60% inhibited by diphenyl iodonium (DPI), stimulated significantly by ubiquinone analogues, and unaffected by metal ions. Production of reactive oxygen species (ROS) in intact cells was not affected by addition of rotenone with proline and malate as substrates; however, addition of rotenone inhibited 41% ROS production with succinate as substrate. These results suggest that complex I is not involved in production of ROS and that succinate-linked reversed electron transport occurs in trypanosome mitochondria. Superoxide formation in mitochondria with NADH as substrate was stimulated by antimycin A but was unaffected by myxothiazol plus stigmatellin, indicating that bc(1) complex is not a source of superoxide. DPI and fumarate inhibited by 68 and 36%, respectively, the rate of superoxide production with NADH as substrate. Addition of both fumarate and DPI blocked 70% superoxide production in mitochondria, a total inhibition similar to that observed with DPI addition alone. These results suggest that the rotenone-insensitive NADH dehydrogenase in addition to NADH fumarate reductase is a potential source of superoxide production in procyclic trypanosome mitochondria.

Topics: Animals; Anti-Bacterial Agents; Antimycin A; Biphenyl Compounds; Fumarates; Malates; Methacrylates; Mitochondria; NAD; NADH Dehydrogenase; Onium Compounds; Polyenes; Proline; Rotenone; Substrate Specificity; Succinic Acid; Superoxides; Thiazoles; Trypanosoma brucei brucei; Ubiquinone; Uncoupling Agents

Following addition of myxothiazol to antimycin-treated chromatophores from Rhodobacter sphaeroides poised at an ambient redox potential (E(h)) of approximately 300 mV, the amplitude of the flash-induced cytochrome c(1) oxidation in the ms range increased, indicating a decrease in the availability of electrons from the immediate donor to c(1), the Rieske iron-sulfur protein (ISP). Because the effect was seen only over the limited E(h) range, we conclude that it is due to a decrease in the apparent midpoint redox potential (E(m)) of the ISP by about 40 mV on addition of myxothiazol. This is in line with the change in E(m) previously seen in direct redox titrations. Our results show that the reduced ISP binds with quinone at the Q(o) site with a higher affinity than does the oxidized ISP. The displacement of ubiquinone by myxothiazol leads to elimination of this preferential binding of the ISP reduced form and results in a shift in the midpoint potential of ISP to a more negative value. A simple hypothesis to explain this effect is that myxothiazol prevents formation of hydrogen bond of ubiquinone with the reduced ISP. We conclude that all Q(o) site occupants (ubiquinone, UHDBT, stigmatellin) that form hydrogen bonds with the reduced ISP shift the apparent E(m) of the ISP in the same direction to more positive values. Inhibitors that bind in the domain of the Q(o) site proximal to heme b(L) (myxothiazol, MOA-stilbene) and displace ubiquinone from the site cause a decrease in E(m) of ISP. We present a new formalism for treatment of the relation between E(m) change and the binding constants involved, which simplifies analysis. Using this formalism, we estimated that binding free energies for hydrogen bond formation with the Q(o) site occupant, range from the largest value of approximately 23 kJ mol(-1) in the presence of stigmatellin (appropriate for the buried hydrogen bond shown by structures), to a value of approximately 3.5 kJ mol(-1) in the native complex. We discuss this range of values in the context of a model in which the native structure constrains the interaction of ISP with the Q(o) site occupant so as to favor dissociation and the faster kinetics of unbinding necessary for rapid turnover.

Topics: Binding Sites; Cytochrome c Group; Cytochromes c1; Cytochromes c2; Electron Transport; Electron Transport Complex III; Iron-Sulfur Proteins; Kinetics; Methacrylates; Models, Chemical; Oxidation-Reduction; Photolysis; Rhodobacter sphaeroides; Spectrophotometry; Thiazoles; Ubiquinone

The steady-state activity of the two quinol-oxidizing pathways of Acanthamoeba castellanii mitochondria, the phosphorylating cytochrome pathway (i.e. the benzohydroxamate(BHAM)-resistant respiration in state 3) and the alternative oxidase (i.e. the KCN-resistant respiration), is shown to be fixed by ubiquinone (Q) pool redox state independently of the reducing substrate (succinate or exogenous reduced nicotinamide adenine dinucleotide (NADH)), indicating that the active Q pool is homogenous. For both pathways, activity increases with the Q reduction level (up to 80%). However, the cytochrome pathway respiration partially inhibited (about 50%) by myxothiazol decreases when the Q reduction level increases above 80%. The decrease can be explained by the Q cycle mechanism of complex III. It is also shown that BHAM has an influence on the relationship between the rate of ADP phosphorylation and the Q reduction level when alternative oxidase is active, and that KCN has an influence on the relationship between the alternative oxidase activity and the Q reduction level. These unexpected effects of BHAM and KCN observed at a given Q reduction level are likely due to functional connections between the two pathways activities or to protein-protein interaction.

Topics: Acanthamoeba; Animals; Cytochromes; Electron Transport; Electron Transport Complex III; Kinetics; Methacrylates; Mitochondria; Mitochondrial Proteins; NAD; Oxidation-Reduction; Oxidoreductases; Plant Proteins; 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

1. Our previous finding that copper ions oxidize nitroxyl anion released from Angeli's salt to nitric oxide prompted us to examine if copper-containing enzymes shared this property. 2. The copper-containing enzyme, tyrosinase, which catalyses the hydroxylation of monophenols to diphenols and the subsequent oxidation of these to the respective unstable quinone, failed to generate nitric oxide from Angeli's salt by itself, but did so in the presence of tyrosine. 3. L-DOPA, the initial product of the reaction of tyrosinase with tyrosine, was not the active species, since it failed to generate nitric oxide from Angeli's salt. Nevertheless, L-DOPA and two other substrates, namely, catechol and tyramine did produce nitric oxide from Angeli's salt in the presence of tyrosinase, suggesting involvement of the respective unstable quinones. In support, we found that 1,4-benzoquinone produced a powerful nitric oxide signal from Angeli's salt. 4. Coenzyme Q(o), an analogue of ubiquinone, failed to generate nitric oxide from Angeli's salt by itself, but produced a powerful signal in the presence of its mitochondrial complex III cofactor, ferricytochrome c. 5. Experiments conducted on rat aortic rings with the mitochondrial complex III inhibitor, myxothiazol, to determine if this pathway was responsible for the vascular conversion of nitroxyl to nitric oxide were equivocal: relaxation to Angeli's salt was inhibited but so too was that to unrelated relaxants. 6. Thus, certain quinones oxidize nitroxyl to nitric oxide. Further work is required to determine if endogenous quinones contribute to the relaxant actions of nitroxyl donors such as Angeli's salt.

Topics: Animals; Aorta, Thoracic; Cytochrome c Group; Electron Transport Complex III; In Vitro Techniques; Male; Methacrylates; Monophenol Monooxygenase; Muscle Relaxation; Muscle, Smooth, Vascular; Nitric Oxide; Nitrites; Nitrogen Oxides; Oxidation-Reduction; Quinones; Rats; Rats, Wistar; Thiazoles; Tyrosine; Ubiquinone; Vasodilation; Vasodilator Agents

The great variability of the human mitochondrial DNA (mtDNA) sequence induces many difficulties in the search for its deleterious mutations. We illustrate these pitfalls by the analysis of the cytochrome b gene of 21 patients affected with a mitochondrial disease. Eighteen different sequence variations were found, five of which were new mutations. Extensive analysis of the cytochrome b gene of 146 controls found 20 supplementary mutations, thus further demonstrating the high variability of the cytochrome b sequence. We fully evaluated the functional relevance of 36 of these 38 mutations using indirect criteria such as the nature of the mutation, its frequency in controls, or the phylogenetic conservation of the mutated amino acid. When appropriate, the mtDNA haplotype, the heteroplasmic state of the mutation, its tissue distribution or its familial transmission were also assessed. The molecular consequences of the mutations, which appeared possibly deleterious in that first step of evaluation, were evaluated on the complex III enzymological properties and protein composition using specific antibodies that we have generated against four of its subunits. Two original deleterious mutations were found in the group of seven patients with overt complex III defect. Both mutations (G15150A (W135X) and T15197C (S151P)) were heteroplasmic and restricted to muscle. They had significant consequences on the complex III structure. In contrast, only two homoplasmic missense mutations with dubious clinical relevance were found in the patients without overt complex III defect.

Topics: Amino Acid Substitution; Antimycin A; Blotting, Western; Cytochrome b Group; DNA Mutational Analysis; DNA, Mitochondrial; Electron Transport Complex III; Gene Frequency; Genetic Variation; Haplotypes; Humans; Methacrylates; Mitochondrial Myopathies; Mutation; Point Mutation; Thiazoles; Ubiquinone

Atovaquone represents a class of antimicrobial agents with a broad-spectrum activity against various parasitic infections, including malaria, toxoplasmosis and Pneumocystis pneumonia. In malaria parasites, atovaquone inhibits mitochondrial electron transport at the level of the cytochrome bc1 complex and collapses mitochondrial membrane potential. In addition, this drug is unique in being selectively toxic to parasite mitochondria without affecting the host mitochondrial functions. A better understanding of the structural basis for the selective toxicity of atovaquone could help in designing drugs against infections caused by mitochondria-containing parasites. To that end, we derived nine independent atovaquone-resistant malaria parasite lines by suboptimal treatment of mice infected with Plasmodium yoelii; these mutants exhibited resistance to atovaquone-mediated collapse of mitochondrial membrane potential as well as inhibition of electron transport. The mutants were also resistant to the synergistic effects of atovaquone/ proguanil combination. Sequencing of the mitochondrially encoded cytochrome b gene placed these mutants into four categories, three with single amino acid changes and one with two adjacent amino acid changes. Of the 12 nucleotide changes seen in the nine independently derived mutants 11 replaced A:T basepairs with G:C basepairs, possibly because of reactive oxygen species resulting from atovaquone treatment. Visualization of the resistance-conferring amino acid positions on the recently solved crystal structure of the vertebrate cytochrome bc1 complex revealed a discrete cavity in which subtle variations in hydrophobicity and volume of the amino acid side-chains may determine atovaquone-binding affinity, and thereby selective toxicity. These structural insights may prove useful in designing agents that selectively affect cytochrome bc1 functions in a wide range of eukaryotic pathogens.

Topics: Amino Acid Sequence; Animals; Antimalarials; Atovaquone; Base Sequence; Chickens; Cytochrome b Group; DNA, Mitochondrial; Drug Resistance; Electron Transport; Membrane Potentials; Methacrylates; Mice; Mice, Inbred BALB C; Mitochondria; Models, Molecular; Molecular Sequence Data; Naphthoquinones; Plasmodium yoelii; Sequence Analysis, DNA; Thiazoles; Ubiquinone

We determined the sites of artificial electron transfer onto 2-nitrosofluorene (NOF), a metabolite of carcinogenic 2- acetylaminofluorene in mitochondria and isolated cytochrome bc1 complex. NOF-induced O2 consumption in mitochondria was sensitive to antimycin A, but insensitive to myxothiazol. In the isolated cytochrome bc1 complex, NOF induced rapid MOA-stilbene-insensitive reoxidation of cytochrome b, whereas in the presence of antimycin A, reoxidation was very slow. The corresponding hydroxylamine, N-hydroxy-2-aminofluorene (N-OH-AF), reduced cytochrome b specifically through center N of the cytochrome bc1 complex. We conclude that NOF and N-OH-AF bind to center N of the cytochrome bc1 complex and act as electron acceptor and donor, respectively. The N-OH-AF/NOF interconversion is considered to be involved in the cytotoxicity of 2-acetylaminofluorene in vivo.

Topics: Animals; Antimycin A; Cytochrome b Group; Dithionite; Electron Transport Complex III; Fluorenes; Male; Methacrylates; Mitochondria, Liver; Nitroso Compounds; Oxidation-Reduction; Oxygen Consumption; Potassium Cyanide; Rats; Rats, Wistar; Stilbenes; Tetramethylphenylenediamine; Thiazoles; Ubiquinone; Uncoupling Agents

Topics: Animals; Fatty Acid Desaturases; In Vitro Techniques; Methacrylates; Mitochondria, Heart; NAD; Oxidation-Reduction; Palmitoyl Coenzyme A; Rats; 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

The dependence of electron flux through quinone-reducing and quinol-oxidizing pathways on the redox state of the ubiquinone (Q) pool was investigated in plant mitochondria isolated from potato (Solanum tuberosum cv. Bintje, fresh tissue and callus), sweet potato (Ipomoea batatas) and Arum italicum. We have determined the redox state of the Q pool with two different methods, the Q-electrode and Q-extraction techniques. Although results from the two techniques agree well, in all tissues tested (with the exception of fresh potato) an inactive pool of QH2 was detected by the extraction technique that was not observed with the electrode. In potato callus mitochondria, an inactive Q pool was also found. An advantage of the extraction method is that it permits determination of the Q redox state in the presence of substances that interfere with the Q-electrode, such as benzohydroxamate and NADH. We have studied the relation between rate and Q redox state for both quinol-oxidizing and quinone-reducing pathways under a variety of metabolic conditions including state 3, state 4, in the presence of myxothiazol, and benzohydroxamate. Under state 4 conditions or in the presence of myxothiazol, a non-linear dependence of the rate of respiration on the Q-redox state was observed in potato callus mitochondria and in sweet potato mitochondria. The addition of benzohydroxamate, under state 4 conditions, removed this non-linearity confirming that it is due to activity of the cyanide-resistant pathway. The relation between rate and Q redox state for the external NADH dehydrogenase in potato callus mitochondria was found to differ from that of succinate dehydrogenase. It is suggested that the oxidation of cytoplasmic NADH in vivo uses the cyanide-resistant pathway more than the pathway involving the oxidation of succinate. A model is used to predict the kinetic behaviour of the respiratory network. It is shown that titrations with inhibitors of the alternative oxidase cannot be used to demonstrate a pure overflow function of the alternative oxidase.

Topics: Benzoquinones; Electron Transport; Hydroquinones; Kinetics; Methacrylates; Mitochondria; NAD; NADH Dehydrogenase; Oxidation-Reduction; Plants; Solanum tuberosum; Succinate Dehydrogenase; Succinates; Succinic Acid; 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

Based on the inhibitor analysis data, it has been assumed that the Q-cycle plays a role in the cyano-resistant malate oxidation induced by menadione (90 microM) in rat liver mitochondria. The extent of involvement of Q-cycle transmitters in the cyano-resistant respiration of mitochondria is determined by the mode of the electron supply into the Q-cycle. In the presence of dicumarol, i.e., under conditions when CoQ and menadione are reduced by NADH-quinone reductase, the bulk of the electrons pass through the o-center of the Q-cycle. Myxothiazole inhibits the respiration by 70-80%, while antimycin--by only 20-30%. In the presence of myxothiazole and antimycin menadione oxidizes cytochrome b. In the presence of rotenone, when menadione is reduced by DT-diaphorase, the rate of cyano-resistant respiration decreases approximately twofold; its sensitivity towards myxothiazole and antimycin drops down to 40%. In the absence of rotenone and dicumarol the Q-cycle does not participate in the cyano-resistant respiration which under these conditions is insensitive either to myxothiazole or to antimycin. It is concluded that the mechanism of cyano-resistant respiration changes with an alteration in the rates of quinones K3 and CoQ reduction. The mechanism of cyano-resistant respiration is also controlled by the medium tonicity. A reduction in the medium tonicity decrease the participation of the Q-cycle and, correspondingly, the sensitivity of the cyano-resistant respiration towards myxothiazole and antimycin.

Topics: Animals; Antimycin A; Cyanides; Electron Transport; Malates; Methacrylates; Mitochondria, Liver; NAD(P)H Dehydrogenase (Quinone); Oxidation-Reduction; Rats; Rotenone; Thiazoles; Ubiquinone; Vitamin K

The reduction of duroquinone (DQ) and 2,3-dimethoxy-5-methyl-6-decyl-1,4-benzoquinone (DB) by NADH and ethanol was investigated in intact yeast mitochondria with good respiratory control ratios. In these mitochondria, exogenous NADH is oxidized by the NADH dehydrogenase localized on the outer surface of the inner membrane, whereas the NADH produced by ethanol oxidation in the mitochondrial matrix is oxidized by the NADH dehydrogenase localized on the inner surface of the inner membrane. The reduction of DQ by ethanol was inhibited 86% by myxothiazol; however, the reduction of DQ by NADH was inhibited 18% by myxothiazol, suggesting that protein-protein interactions between the internal (but not the external) NADH: ubiquinone oxidoreductase and ubiquinol:cytochrome c oxidoreductase (the cytochrome bc1 complex) are involved in the reduction of DQ by NADH. The reduction of DQ and DB by NADH and ethanol was also investigated in mutants of yeast lacking cytochrome b, the iron-sulfur protein, and ubiquinone. The reduction of both quinone analogues by exogenous NADH was reduced to levels that were 10 to 20% of those observed in wild-type mitochondria; however, the rate of their reduction by ethanol in the mutants was equal to or greater than that observed in the wild-type mitochondria. Furthermore, the reduction of DQ in the cytochrome b and iron-sulfur protein lacking mitochondria was myxothiazol sensitive, suggesting that neither of these proteins is an essential binding site for myxothiazol. The mitochondria from the three mutants also contained significant amounts of antimycin- and myxothiazol-insensitive NADH:cytochrome c reductase activity, but had no detectable succinate:cytochrome c reductase activity. These results suggest that the mutants lacking a functional cytochrome bc1 complex have adapted to oxidize NADH.

Topics: Antimycin A; Cytochrome c Group; Electron Transport; Electron Transport Complex III; Ethanol; Kinetics; Methacrylates; Mitochondria; Models, Biological; NAD; NAD(P)H Dehydrogenase (Quinone); Quinones; Saccharomyces cerevisiae; Thiazoles; Ubiquinone

We have investigated the role of the Coenzyme Q pool in glycerol-3-phosphate oxidation in hamster brown adipose tissue mitochondria. Antimycin A and myxothiazol inhibit glycerol-3-phosphate cytochrome c oxidoreductase in a sigmoidal fashion, indicating that CoQ behaves as a homogeneous pool between glycerol-3-phosphate dehydrogenase and complex III. The inhibition of ubiquinol cytochrome c reductase is linear at low concentrations of both inhibitors, indicating that sigmoidicity of antimycin A and myxothiazol inhibition is not a direct property of antimycin A and myxothiazol binding. Glycerol-3-phosphate cytochrome c oxidoreductase is strongly stimulated by added CoQ3, indicating that endogenous CoQ is not saturating. Application of the pool equation for nonsaturating ubiquinone allows calculation of the Km for endogenous CoQ of glycerol-3-phosphate dehydrogenase of 3.14 mM. The results of this investigations reveal that CoQ behaves as a homogeneous pool between glycerol-3-phosphate dehydrogenase and complex III in brown adipose tissue mitochondria; moreover, its concentration is far below saturation for maximal electron transfer activity in comparison with other branches of the respiratory chain connected with the CoQ pool. HPLC analysis revealed a lower amount of CoQ in brown adipose mitochondria (0.752 nmol/mg protein) in comparison with mitochondria from other tissues and the presence of both CoQ9 and CoQ10.

Topics: Adipose Tissue, Brown; Animals; Antimycin A; Carbohydrate Dehydrogenases; Cricetinae; Electron Transport Complex III; Glycerolphosphate Dehydrogenase; Glycerophosphates; Mesocricetus; Methacrylates; Mitochondria; Oxidation-Reduction; Palmitoyl Coenzyme A; 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 steady-state reduction of exogenous ubiquinone-2 by duroquinol as catalysed by the ubiquinol: cytochrome c oxidoreductase was studied in bovine heart mitoplasts. The reduction of ubiquinone-2 by duroquinol proceeds both in the absence of inhibitors of the enzyme, in the presence of outside inhibitors, e.g., myxothiazol, and in the presence of inside inhibitors, e.g., antimycin, but not in the presence of both inside and outside inhibitors. It is concluded that both the Qin-binding domain and the Qout-binding domain may independently catalyse this reaction. The rate of the reduction of ubiquinone-2 by duroquinol via the Qin-binding domain is dependent on the type of outside inhibitor used. The maximal rate obtained for the reduction of ubiquinone-2 by DQH2 via the Qout-binding domain, measured in the presence of antimycin, is similar to that catalysed by the Qin-binding domain of the non-inhibited enzyme and depends on the redox state of the high-potential electron carriers of the respiratory chain. The reduction of ubiquinone-2 by DQH2 via the Qin-binding domain can be described by a mechanism in which duroquinol reduces the enzyme, upon which the reduced enzyme is rapidly oxidized by ubiquinone-2 yielding ubiquinol-2. By determination of the initial rate under various conditions and simulation of the time course of reduction of ubiquinone-2 using the integrated form of the steady-state rate equation the values of the various kinetic constants were calculated. During the course of reduction of ubiquinone-2 by duroquinol in the presence of outside inhibitors only cytochrome b-562 becomes reduced. At all stages during the reaction, cytochrome b-562 is in equilibrium with the redox potential of the ubiquinone-2/ubiquinol-2 couple but not with that of the duroquinone/duroquinol couple. At low pH values, cytochrome b-562 is reduced in a single phase; at high pH separate reduction phases are observed. In the absence of inhibitors three reduction phases of cytochrome b-562 are discernible at low pH values and two at high pH values. In the presence of antimyin cytochrome b becomes reduced in two phases. Cytochrome b-562 is reduced in the first phase and cytochrome b-566 in the second phase after substantial reduction of ubiquinone-2 to ubiquinol-2 has occurred. In ubiquinone-10 depleted preparations, titration of cytochrome b-562, in the presence of myxothiazol, with the duroquinone/duroquinol redox couple yields a value of napp = 2, both at low and high pH.(

Topics: Animals; Antimycin A; Benzoquinones; Binding Sites; Cattle; Cytochrome b Group; Electron Transport; Electron Transport Complex III; Hydrogen-Ion Concentration; Hydroquinones; Methacrylates; Myocardium; Oxidation-Reduction; Thiazoles; Ubiquinone

EPR characteristics of cytochrome c1, cytochromes b-565 and b-562, the iron-sulfur cluster, and an antimycin-sensitive ubisemiquinone radical of purified cytochrome b-c1 complex of Rhodobacter sphaeroides have been studied. The EPR specra of cytochrome c1 shows a signal at g = 3.36 flanked with shoulders. The oxidized form of cytochrome b-562 shows a broad EPR signal at g = 3.49, while oxidized cytochrome b-565 shows a signal at g = 3.76, similar to those of two b cytochromes in the mitochondrial complex. The distribution of cytochromes b-565 and b-562 in the isolated complex is 44 and 56%, respectively. Antimycin and 2,5-dibromo-3-methyl-6-isopropyl-1,4-benzoquinone (DBMIB) have little effect on the g = 3.76 signal, but they cause a slight downfield and upfield shifts of the g = 3.49 signal, respectively. 5-Undecyl-6-hydroxyl-4,7-dioxobenzothiazole (UHDBT) shifts the g = 3.49 signal downfield to g = 3.56 and sharpens the g = 3.76 signal slightly. Myxothiazol causes an upfield shift of both g = 3.49 and g = 3.76 signals. EPR characteristics of the reduced iron-sulfur cluster in bacterial cytochrome b-c1 complex are: gx = 1.8 with a small shoulder at g = 1.76, gy = 1.89 and gz = 2.02, similar to those observed with the mitochondrial enzyme. The gx = 1.8 signal decreased and the shoulder increased concurrently as the redox potential decreased, indicating that the environment of the iron-sulfur cluster is sensitive to the redox state of the complex. UHDBT sharpens the gz and and shifts it downfield from g = 2.02 to 2.03, and shifts gx upfield from g = 1.80 to 1.78. UHDBT also causes an upfield shift of gy but to a much lesser extent compared to the other two signals. Addition of DBMIB causes a downfield shift of the gy from 1.89 to 1.94 and broadens the gx signal with an upfield to g = 1.75. Myxothiazol and antimycin show little effect on the gy and gz signals, but they broaden and shift the gx signal upfield to g = 1.74. However, the myxothiazol effect is partially reversed by UHDBT. An antimycin-sensitive ubisemiquinone radical was detected in the cytochrome b-c1 complex. At pH 8.4, the antimycin-sensitive ubisemiquinone radical has a maximal concentration of 0.66 mol per mol complex at 100 mV.(ABSTRACT TRUNCATED AT 400 WORDS)

Topics: Antimycin A; Coenzymes; Cytochrome b Group; Electron Spin Resonance Spectroscopy; Electron Transport; Electron Transport Complex III; Iron-Sulfur Proteins; Methacrylates; Oxidation-Reduction; Rhodobacter sphaeroides; 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

Mitochondria exhibiting cyanide-resistant respiration were isolated from Hansenula anomala which had been incubated in the presence of antimycin A to induce cyanide-resistant respiration. The cyanide-resistant respiration in isolated mitochondria was not inhibited by antimycin A or myxothiazol, suggesting that the branching of the pathway from the normal cyanide-sensitive pathway takes place at the coenzyme Q level. Analysis of mitochondrial proteins by sodium dodecyl sulfate gel electrophoresis indicated that a 36 kDa protein was induced by antimycin A treatment of the yeast. It is suggested that this protein is a component of the cyanide-resistant respiratory pathway.

Topics: Antifungal Agents; Antimycin A; Centrifugation, Density Gradient; Cyanides; Electrophoresis, Polyacrylamide Gel; Fungal Proteins; Methacrylates; Methionine; Mitochondria; Molecular Weight; Oxidation-Reduction; Oxygen Consumption; Pichia; Sulfur Radioisotopes; Thiazoles; Ubiquinone

The role of subunit VII, the ubiquinone-binding protein of the cytochrome b-c1 complex, in electron transfer reactions was investigated in yeast mitochondria. Preincubation of submitochondrial particles with specific antibody against subunit VII prior to addition of either succinate, NADH, or the reduced form of the decyl analogue of ubiquinol resulted in an approximately 40% increase in the extent of cytochrome c1 reduction compared with controls containing preimmune serum. Addition of antimycin, an inhibitor of center i, to submitochondrial particles resulted in a 21% decrease in the rate and a 36% decrease in the extent of cytochrome c1 reduction by succinate. Preincubation of submitochondrial particles with the antibody against subunit VII prior to addition of antimycin resulted in an increase in both the rate and extent of cytochrome c1 reduction to the levels observed in the control without inhibitor. The addition of myxothiazol (an inhibitor of center o), myxothiazol plus antimycin, or alkyl hydroxynaphthoquinone (an inhibitor analogue of ubiquinone) resulted in an almost complete inhibition in both the rate and extent of cytochrome c1 reduction; however, preincubation with the antibody against subunit VII prior to addition of these inhibitors resulted in a significant increase in cytochrome c1 reduction. These results confirm our previous report (Japa, S., Zhu, Q. S., and Beattie, D. S. (1987) J. Biol. Chem. 262, 5441-5444) that subunit VII is involved in electron transfer reactions at center o of the b-c1 complex. We suggest that the binding of antibody to subunit VII inhibits the transfer of electrons to cytochrome b-566. Consequently, two electrons are transferred to the iron-sulfur protein and cytochrome c1 through an antimycin-insensitive pathway. Moreover, the antibody may change the conformation of subunit VII, such that the myxothiazol and hydroxynaphthoquinone binding sites are partially blocked thus permitting electron flow to cytochrome c1.

Topics: Antibodies, Fungal; Antimycin A; Carrier Proteins; Cytochromes c1; Electron Transport; Electron Transport Complex III; Methacrylates; Mitochondria; Saccharomyces cerevisiae; Thiazoles; Ubiquinone

The interaction of the exogenous quinones, duroquinone (DQ) and the decyl analogue of ubiquinone (DB) with the mitochondrial respiratory chain was studied in both wild-type and a ubiquinone-deficient mutant of yeast. DQ can be reduced directly by NADH dehydrogenase, but cannot be reduced by succinate dehydrogenase in the absence of endogenous ubiquinone. The succinate-driven reduction of DQ can be stimulated by DB in a reaction inhibited 50% by antimycin and 70-80% by the combined use of antimycin and myxothiazol, suggesting that electron transfer occurs via the cytochrome b-c1 complex. Both DQ and DB can effectively mediate the reduction of cytochrome b by the primary dehydrogenases through center o, but their ability to mediate the reduction of cytochrome b through center i is negligible. Two reaction sites for ubiquinol seem to be present at center o: one is independent of endogenous Q6 with a high reaction rate and a high Km; the other is affected by endogenous Q6 and has a low reaction rate and a low Km. By contrast, only one ubiquinol reaction site was observed at center i, where DB appears to compete with endogenous Q6. DB can oxidize most of the pre-reduced cytochrome b, while DQ can oxidize only 50%. On the basis of these data, the possible binding patterns of DB on different Q-reaction sites and the requirement for ubiquinone in the continuous oxidation of DQH are discussed.

Topics: Antimycin A; Benzoquinones; Cytochrome b Group; Electron Transport; Electron Transport Complex III; Kinetics; Methacrylates; Mitochondria; Oxidation-Reduction; Quinones; Saccharomyces cerevisiae; Succinate Dehydrogenase; Succinates; Succinic Acid; Thiazoles; Ubiquinone

The kinetic model of Ragan & Cottingham [(1985) Biochim. Biophys. Acta 811, 13-31] for electron transport through a mobile pool of quinone predicts that, under certain conditions, the normal linear dependence of electron flow on the degree of reduction (or oxidation) of the quinone should no longer be found. These conditions can be met by reconstituted NADH: cytochrome c reductase (Complex I-III from bovine heart) when electron flow is rate-limited by a low concentration of cytochrome c. We show that, in such a system, the dependence of activity (varied by inhibition with rotenone) on the steady-state level of quinone reduction is indeed non-linear and very closely accounted for by the theory.

Topics: Animals; Cattle; Cytochrome c Group; Cytochrome Reductases; Electron Transport; Kinetics; Methacrylates; Models, Chemical; NADH Dehydrogenase; Oxidation-Reduction; Rotenone; 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

Much evidence indicates that superoxide is generated from O2 in a cyanide-sensitive reaction involving a reduced component of complex III of the mitochondrial respiratory chain, particularly when antimycin A is present. Although it is generally believed that ubisemiquinone is the electron donor to O2, little experimental evidence supporting this view has been reported. Experiments with succinate as electron donor in the presence of antimycin A in intact rat heart mitochondria, which contain much superoxide dismutase but little catalase, showed that myxothiazol, which inhibits reduction of the Rieske iron-sulfur center, prevented formation of hydrogen peroxide, determined spectrophotometrically as the H2O2-peroxidase complex. Similarly, depletion of the mitochondria of their cytochrome c also inhibited formation of H2O2, which was restored by addition of cytochrome c. These observations indicate that factors preventing the formation of ubisemiquinone also prevent H2O2 formation. They also exclude ubiquinol, which remains reduced under these conditions, as the reductant of O2. Since cytochrome b also remains fully reduced when myxothiazol is added to succinate- and antimycin A-supplemented mitochondria, reduced cytochrome b may also be excluded as the reductant of O2. These observations, which are consistent with the Q-cycle reactions, by exclusion of other possibilities leave ubisemiquinone as the only reduced electron carrier in complex III capable of reducing O2 to O2-.

Topics: Animals; Antimycin A; Coenzymes; Cytochrome b Group; Cytochrome c Group; Electron Transport; Electron Transport Complex III; Hydrogen Peroxide; In Vitro Techniques; Methacrylates; Mitochondria, Heart; Multienzyme Complexes; NADH, NADPH Oxidoreductases; Oxygen Consumption; Quinone Reductases; Rats; Succinates; Superoxides; 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

Two different bypasses around the antimycin block of electron transport from succinate to cytochrome c via the ubiquinol-cytochrome c oxidoreductase of intact rat liver mitochondria were analyzed, one promoted by N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD) and the other by 2,6-dichlorophenolindophenol (DCIP). Both bypasses are inhibited by myxothiazol, which blocks electron flow from ubiquinol to the Rieske iron-sulfur center, and by 2-hydroxy-3-undecyl-1,4-naphthoquinone, which inhibits electron flow from the iron-sulfur center to cytochrome c1. In the bypass promoted by TMPD its oxidized form (Wurster's blue) acts as an electron acceptor from some reduced component prior to the antimycin block, which by exclusion of other possibilities is ubisemiquinone. In the DCIP bypass its reduced form acts as an electron donor, by reducing ubisemiquinone to ubiquinol; reduced DCIP is regenerated again at the expense of either succinate or ascorbate. The observations described are consistent with and support current models of the Q cycle. Bypasses promoted by artificial electron carriers provide an independent approach to analysis of electron flow through ubiquinol-cytochrome c oxidoreductase.

Topics: 2,6-Dichloroindophenol; Animals; Antimycin A; Coenzymes; Cytochrome b Group; Cytochrome c Group; Electron Transport; Electron Transport Complex III; Methacrylates; Mitochondria, Liver; Multienzyme Complexes; Naphthoquinones; Quinone Reductases; Rats; Succinates; Succinic Acid; Tetramethylphenylenediamine; 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

(1) The kinetics of the reduction by duroquinol of the prosthetic groups of QH2:cytochrome c oxidoreductase and of the formation of ubisemiquinone have been studied using a combination of the freeze-quench technique, low-temperature diffuse-reflectance spectroscopy, EPR and stopped flow. (2) The formation of the antimycin-sensitive ubisemiquinone anion parallels the reduction of both high-potential and low-potential cytochrome b-562. (3) The rates of reduction of both the [2Fe-2S] clusters and cytochromes (c + c1) are pH dependent. There is, however, a pH-dependent discrepancy between their rate of reduction, which can be correlated with the difference in pH dependencies of their midpoint potentials. (4) Lowering the pH or the Q content results in a slower reduction of part of the [2Fe-2S] clusters. It is suggested that one cluster is reduced by a quinol/semiquinone couple and the other by a semiquinone/quinone couple. (5) Myxothiazol inhibits the reduction of the [2Fe-2S] clusters, cytochrome c1 and high-potential cytochrome b-562. (6) The results are consistent with a Q-cycle model describing the pathway of electrons through a dimeric QH2:cytochrome c oxidoreductase.

Topics: Animals; Cattle; Cytochrome b Group; Electron Spin Resonance Spectroscopy; Electron Transport Complex III; Escherichia coli Proteins; Hydrogen-Ion Concentration; Kinetics; Methacrylates; Mitochondria, Heart; Multienzyme Complexes; NADH, NADPH Oxidoreductases; Oxidation-Reduction; Quinone Reductases; Submitochondrial Particles; 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