5-n-undecyl-6-hydroxy-4-7-dioxobenzothiazole and myxothiazol

Topics: Animals; Antimycin A; Cyanates; Electron Transport Complex III; Electron Transport Complex IV; Humans; Methacrylates; Mitochondria; Nitrous Oxide; Polyenes; Polylysine; Thiazoles

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

A detergent-solubilized, three-subunit-containing cytochrome bc1 complex, isolated from the photosynthetic bacterium R. rubrum, has been shown to be highly sensitive to stigmatellin, myxothiazol, antimycin A and UHDBT, four specific inhibitors of these complexes. Oxidation-reduction titrations have allowed the determination of Em values for all the electron-carrying prosthetic groups in the complex. Antimycin A has been shown to produce a red shift in the alpha-band absorbance maximum of one of the cytochrome b hemes in the complex and stigmatellin has been shown to alter both the Em and EPR g-values of the Rieske iron-sulfur protein in the complex. Western blots have revealed antigenic similarities between the cytochrome subunits of the R. rubrum complex and those of the related photosynthetic bacteria, Rb. capsulatus and Rb. sphaeroides. The R. rubrum complex has been incorporated into liposomes. These liposomes exhibit respiratory control and are able to couple electron transfer from quinol to cytochrome c to proton translocation across the liposome membrane in a manner consistent with a Q-cycle mechanism. It can thus be concluded that neither electron transport nor coupled proton translocation by the cytochrome bc1 complex requires more than three subunits in R. rubrum.

Topics: Antimycin A; Blotting, Western; Electron Spin Resonance Spectroscopy; Electron Transport Complex III; Heme; Iron-Sulfur Proteins; Methacrylates; Oxidation-Reduction; Polyenes; Protons; Rhodospirillum rubrum; Thiazoles

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

Although the function of the Rieske iron-sulfur protein is generally understood, little is known of how the structure of this protein supports its mechanistic role in electron transfer in the cytochrome bc1 complex. To better understand the structural basis of iron-sulfur protein function, we have undertaken a mutational analysis of the gene encoding this protein and initially isolated five temperature-sensitive iron-sulfur protein mutants (Beckmann, J. D., Ljungdahl, P. O., and Trumpower, B. L. (1989) J. Biol. Chem. 264, 3713-3722). Each of the five ts-rip1- mutants exhibited pleiotropic effects. Although the mutant iron-sulfur proteins manifest several in vitro phenotypes in common, each exhibited unique characteristics. All of the ts-rip1- mutations resulted in membranes with decreased ubiquinol-cytochrome c oxidoreductase activities and decreased thermostability compared to membranes containing wild type iron-sulfur protein. All of the mutations conferred slight but significant resistance to the respiratory inhibitor myxothiazol, and one mutant was hypersensitive to inhibition by UHDBT, a structural analog of ubiquinone. In addition, one of the mutations completely blocks post-translational processing of the iron-sulfur protein, leading to accumulation of pre-iron-sulfur protein in mitochondrial membranes at nonpermissive temperatures. Finally, a mutation 12-amino acid residues away from the carboxyl terminus (203S) results in an extremely unstable protein. This region of the protein may be essential in blocking degradation of pre-iron-sulfur protein by cytoplasmic proteases as the protein is imported into the mitochondria, or may be a "degradation signal," which tags the iron-sulfur protein for turnover.

Topics: Antimycin A; Blotting, Western; DNA Mutational Analysis; Electron Spin Resonance Spectroscopy; Electron Transport; Electron Transport Complex III; Intracellular Membranes; Iron-Sulfur Proteins; Membrane Proteins; Metalloproteins; Methacrylates; Mitochondria; NADH Dehydrogenase; Plasmids; Saccharomyces cerevisiae; Spectrum Analysis; Structure-Activity Relationship; Temperature; Thiazoles

We have examined the effects of eight inhibitors of the bovine-heart mitochondrial Complex III on the catalytic activity of the analogous complex from yeast mitochondria. All eight compounds were inhibitory, with potent inhibition being obtained with antimycin, myxothiazol and UHDBT (5-N-undecyl-6-hydroxy-4,7-dioxobenzothiazole). These three inhibitors, and also funiculosin, have been further studied by characterizing their effects on the visible absorbance, magnetic circular dichroism and EPR spectra of the complex and also on the potentiometric properties of the individual metal centers present in the complex. All four inhibitors had little or no effect on either the absorbance or magnetic circular dichroism spectra. Funiculosin produced a change in the EPR lineshape of the iron-sulfur cluster; EPR spectra recorded at 12 K also revealed complete reduction of cytochrome b-562 by ascorbate. UHDBT also changed the lineshape of the iron-sulfur cluster and this change could be partially reversed by myxothiazol. Neither antimycin nor myxothiazol affected the iron-sulfur cluster and produced only small changes in the EPR absorption envelope of the b cytochromes. Both funiculosin and UHDBT raised the midpoint potential of the iron-sulfur cluster, by about 150 and 70 mV, respectively. Only UHDBT changed the potential of c1, lowering it by about 30 mV. Funiculosin raised the potential of b-562 by about 30 mV, while myxothiazol had no effect; the other two compounds produced only small changes. All four compounds had only small effects on the midpoint potential of b-566. The relative contributions of the two b cytochromes to the magnetic circular dichroism amplitudes could be changed by the addition of inhibitors, even though the absolute magnetic circular dichroism spectra of oxidized and reduced complex were unaffected.

Topics: Anthraquinones; Antimycin A; Circular Dichroism; Cytochrome b Group; Cytochrome c Group; Electron Spin Resonance Spectroscopy; Electron Transport Complex III; Iron-Sulfur Proteins; Methacrylates; Multienzyme Complexes; NADH, NADPH Oxidoreductases; Oxidation-Reduction; Quinone Reductases; Saccharomyces cerevisiae; Spectrophotometry; Thiazoles

The QH2:cytochrome c oxidoreductase activity of the isolated bovine heart cytochrome b-c1 complex resolved into monomeric and dimeric form was titrated with three different inhibitors of electron transfer, antimycin, myxothiazol, and 5-n-undecyl-6-hydroxy-4,7-dioxobenzothiazole (UHDBT). In all cases one inhibitor molecule per cytochrome c1 was found necessary to block completely the activity of both molecular forms of the enzyme. The antimycin-sensitive cytochrome c reduction catalyzed by the b-c1 complex was also studied as a function of increasing concentrations of either cytochrome c or quinol. Double-reciprocal plots of the activity of the monomeric enzyme were found linear either when the concentration of cytochrome c or of quinol derivatives, 2,3-dimetoxy-5-methyl-6-decyl-1,4-benzoquinol (DBH), and 2-methyl-3-undecyl-1,4-naphthoquinol (UNH), was changed. Cytochrome c reductase activity of the dimeric b-c1 complex also showed a linear Lineweaver-Burk plot as a function of cytochrome c concentrations. In contrast to the monomeric enzyme, however, dimers of the b-c1 complex express a clear nonlinear kinetic behavior toward quinol derivatives, with two apparent Km values differing approximately by one order of magnitude (about 3-4 and about 20-30 microM). At saturating quinol concentrations the activity of the dimeric enzyme becomes two to three times higher than that of monomers. The nonlinear kinetic plots were found to be the same at different temperatures and different cytochrome c concentrations. The data suggest that although the monomer of the b-c1 complex appears to be the functional unit of the enzyme, the dimer is more active. A regulatory role of the dimerization process resulting in an increase of the electrons flux through the enzyme is postulated.

Topics: Animals; Antimycin A; Cattle; Cytochrome c Group; Electron Transport Complex III; Hydroquinones; Kinetics; Macromolecular Substances; Methacrylates; Mitochondria, Heart; Multienzyme Complexes; NADH, NADPH Oxidoreductases; Quinone Reductases; Thiazoles

Myxothiazol inhibited the electron transport in the cytochrome b/c segment of membrane particles from Pseudomonas cichorii. A residual NADH-oxidation due to the presence of an alternative pathway via cytochrome o (Em, 7 = +250 mV) was sensitive to the quinone analog 5-undecyl-6-hydroxy-4,7-dioxobenzothiazole (UHDBT). This latter inhibitor was equally effective in blocking the linear respiratory chain of Pseudomonas aptata, a strain deficient in cytochromes of c type and Rieske iron-sulphur centre. The analysis of the oxido-reduction kinetic patterns of cytochromes indicated that, among the b type haems present in P. aptata, only cyt. o could be reduced by ubiquinol-1 in a reaction insensitive to both antimycin A and myxothiazol but inhibited by UHDBT. This latter finding has been correlated to the fact that P. aptata exhibits a defective b/c complex. In membranes from P. cichorii, in which the absorption maximum of dithionite reduced cytochrome(s) b shifted by 2-3 nm in the presence of antimycin A and/or myxothiazol, the electron flow through the b/c oxidoreductase complex has tentatively been arranged in a proton motive "Q-cycle" like mechanism.

Topics: Antifungal Agents; Fluorescence; Kinetics; Methacrylates; Oxygen Consumption; Pseudomonas; Species Specificity; Thiazoles