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Raul Covian - One of the best experts on this subject based on the ideXlab platform.
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asymmetric binding of stigmatellin to the dimeric paracoccus denitrificans bc1 complex evidence for anti cooperative ubiquinol oxidation and communication between center p ubiquinol oxidation sites
Journal of Biological Chemistry, 2007Co-Authors: Raul Covian, Thomas Kleinschroth, Bernd LudwigAbstract:Abstract We have investigated the mechanism responsible for half-of-the-sites activity in the dimeric cytochrome bc1 complex from Paracoccus denitrificans by characterizing the kinetics of inhibitor binding to the ubiquinol oxidation site at center P. Both Myxothiazol and stigmatellin induced a 2–3 nm shift of the visible absorbance spectrum of the bL heme. The shift generated by Myxothiazol was symmetric, with monophasic kinetics that indicate equal binding of this inhibitor to both center P sites. In contrast, stigmatellin generated an asymmetric shift in the bL spectrum, with biphasic kinetics in which each phase contributed approximately half of the total magnitude of the spectral change. The faster binding phase corresponded to a more symmetrical shift of the bL spectrum relative to the slower binding phase, indicating that approximately half of the center P sites bound stigmatellin more slowly and in a different position relative to the bL heme, generating a different effect on its electronic environment. Significantly, the slow stigmatellin binding phase was lost as the inhibitor concentration was increased. This implies that a conformational change is transmitted from one center P site in the dimer to the other upon stigmatellin binding to one monomer, rendering the second site less accessible to the inhibitor. Because the position that stigmatellin occupies at center P is considered to be analogous to that of the quinol substrate at the moment of electron transfer, these results indicate that the productive enzyme-substrate configuration is prevented from occurring in both monomers simultaneously.
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regulatory interactions between ubiquinol oxidation and ubiquinone reduction sites in the dimeric cytochrome bc1 complex
Journal of Biological Chemistry, 2006Co-Authors: Raul Covian, Bernard L. TrumpowerAbstract:We have obtained evidence for conformational communication between ubiquinol oxidation (center P) and ubiquinone reduction (center N) sites of the yeast bc1 complex dimer by analyzing antimycin binding and heme bH reduction at center N in the presence of different center P inhibitors. When stigmatellin was occupying center P, concentration-dependent binding of antimycin occurred only to half of the center N sites. The remaining half of the bc1 complex bound antimycin with a slower rate that was independent of inhibitor concentration, indicating that a slow conformational change needed to occur before half of the enzyme could bind antimycin. In contrast, under conditions where the Rieske protein was not fixed proximal to heme bL at center P, all center N sites bound antimycin with fast and concentration-dependent kinetics. Additionally, the extent of fast cytochrome b reduction by menaquinol through center N in the presence of stigmatellin was approximately half of that observed when Myxothiazol was bound at center P. The reduction kinetics of the bH heme by decylubiquinol in the presence of stigmatellin or Myxothiazol were also consistent with a model in which fixation of the Rieske protein close to heme bL in both monomers allows rapid binding of ligands only to one center N. Decylubiquinol at high concentrations was able to abolish the biphasic binding of antimycin in the presence of stigmatellin but did not slow down antimycin binding rates. These results are discussed in terms of half-of-the-sites activity of the dimeric bc1 complex.
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tight binding of inhibitors to bovine bc1 complex is independent of the rieske protein redox state consequences for semiquinone stabilization in the quinol oxidation site
Journal of Biological Chemistry, 2002Co-Authors: Raul Covian, Juan Pablo Pardo, Rafael MorenosanchezAbstract:To determine the effect of the redox state of the Rieske protein on ligand binding to the quinol oxidation site of the bc(1) complex, we measured the binding rate constants (k(1)) for stigmatellin and Myxothiazol, at different concentrations of decylbenzoquinone or decylbenzoquinol, in the bovine bc(1) complex with the Rieske protein in the oxidized or reduced state. Stigmatellin and Myxothiazol bound tightly and competitively with respect to quinone or quinol, independently of the redox state of the Rieske protein. In the oxidized bc(1) complex, the k(1) values for stigmatellin ( approximately 2.6 x 10(6) m(-1)s(-1)) and Myxothiazol ( approximately 8 x 10(5) m(-1)s(-1)), and the dissociation constant (K(d)) for quinone, were similar between pH 6.5 and 9, indicating that ligand binding is independent of the protonation state of histidine 161 of the Rieske protein (pK(a) approximately 7.6). Reduction of the Rieske protein increased the k(1) value for stigmatellin and decreased the K(d) value for quinone by 50%, without modifying the k(1) for Myxothiazol. These results indicate that reduction of the Rieske protein and protonation of histidine 161 do not induce a strong stabilization of ligand binding to the quinol oxidation site, as assumed in models that propose the existence of a highly stabilized semiquinone as a reaction intermediate during quinol oxidation.
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Tight binding of inhibitors to bovine bc1 complex is independent of the Rieske protein redox state. Consequences for semiquinone stabilization in the quinol oxidation site.
The Journal of biological chemistry, 2002Co-Authors: Raul Covian, Juan Pablo Pardo, Rafael Moreno-sánchezAbstract:Abstract To determine the effect of the redox state of the Rieske protein on ligand binding to the quinol oxidation site of the bc 1 complex, we measured the binding rate constants (k 1) for stigmatellin and Myxothiazol, at different concentrations of decylbenzoquinone or decylbenzoquinol, in the bovine bc 1 complex with the Rieske protein in the oxidized or reduced state. Stigmatellin and Myxothiazol bound tightly and competitively with respect to quinone or quinol, independently of the redox state of the Rieske protein. In the oxidized bc 1 complex, thek 1 values for stigmatellin (∼2.6 × 106 m −1s−1) and Myxothiazol (∼8 × 105 m −1s−1), and the dissociation constant (K d) for quinone, were similar between pH 6.5 and 9, indicating that ligand binding is independent of the protonation state of histidine 161 of the Rieske protein (pK a ∼7.6). Reduction of the Rieske protein increased the k 1 value for stigmatellin and decreased the K d value for quinone by 50%, without modifying the k 1 for Myxothiazol. These results indicate that reduction of the Rieske protein and protonation of histidine 161 do not induce a strong stabilization of ligand binding to the quinol oxidation site, as assumed in models that propose the existence of a highly stabilized semiquinone as a reaction intermediate during quinol oxidation.
Sergei A. Dikanov - One of the best experts on this subject based on the ideXlab platform.
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The interaction of the Rieske iron-sulfur protein with occupants of the Qo-site of the bc1 complex, probed by electron spin echo envelope modulation
Archaea, 2015Co-Authors: Taketoshi Uzawa, Toshio Iwasaki, Sergei A. DikanovAbstract:The bifurcated reaction at the Qo-site of the bc1 com-plex provides the mechanistic basis of the proton pump-ing activity through which the complex conserves redox energy in the proton gradient. Structural information about the binding of quinone at the site is lacking, be-cause the site is vacant in crystals of the native com-plexes. We now report the first structural characteriza-tion of the interaction of the native quinone occupant with the Rieske iron-sulfur protein in the bc1 complex of Rhodobacter sphaeroides, using high resolution EPR. We have compared the binding configuration in the presence of quinone with the known structures for the complex with stigmatellin and Myxothiazol. We have shown by using EPR and orientation-selective electron spin echo envelope modulation (ESEEM) measurement
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The interaction of the Rieske iron-sulfur protein with occupants of the Qo-site of the bc1 complex, probed by electron spin echo envelope modulation.
The Journal of biological chemistry, 2001Co-Authors: Rimma I. Samoilova, Antony R Crofts, Derrick R. J. Kolling, Taketoshi Uzawa, Toshio Iwasaki, Sergei A. DikanovAbstract:The bifurcated reaction at the Q(o)-site of the bc(1) complex provides the mechanistic basis of the proton pumping activity through which the complex conserves redox energy in the proton gradient. Structural information about the binding of quinone at the site is lacking, because the site is vacant in crystals of the native complexes. We now report the first structural characterization of the interaction of the native quinone occupant with the Rieske iron-sulfur protein in the bc(1) complex of Rhodobacter sphaeroides, using high resolution EPR. We have compared the binding configuration in the presence of quinone with the known structures for the complex with stigmatellin and Myxothiazol. We have shown by using EPR and orientation-selective electron spin echo envelope modulation (ESEEM) measurements of the iron-sulfur protein that when quinone is present in the site, the isotropic hyperfine constant of one of the N(delta) atoms of a liganding histidine of the [2Fe-2S] cluster is similar to that observed when stigmatellin is present and different from the configuration in the presence of Myxothiazol. The spectra also show complementary differences in nitrogen quadrupole splittings in some orientations. We suggest that the EPR characteristics, the ESEEM spectra, and the hyperfine couplings reflect a similar interaction between the iron-sulfur protein and the quinone or stigmatellin and that the N(delta) involved is that of a histidine (equivalent to His-161 in the chicken mitochondrial complex) that forms both a ligand to the cluster and a hydrogen bond with a carbonyl oxygen atom of the Q(o)-site occupant.
Rolf Müller - One of the best experts on this subject based on the ideXlab platform.
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Reconstitution of the Myxothiazol Biosynthetic Gene Cluster by Red/ET Recombination and Heterologous Expression in Myxococcus xanthus
Applied and Environmental Microbiology, 2006Co-Authors: Olena Perlova, Silvia Kuhlmann, Daniel Krug, Youming Zhang, Jun Fu, A. Francis Stewart, Rolf MüllerAbstract:Although many secondary metabolites exhibiting important pharmaceutical and agrochemical activities have been isolated from myxobacteria, most of these microorganisms remain difficult to handle genetically. To utilize their metabolic potential, heterologous expression methodologies are currently being developed. Here, the Red/ET recombination technology was used to perform all required gene cluster engineering steps in Escherichia coli prior to the transfer into the chromosome of the heterologous host. We describe the integration of the complete 57-kbp Myxothiazol biosynthetic gene cluster reconstituted from two cosmids from a cosmid library of the myxobacterium Stigmatella aurantiaca DW4-3/1 into the chromosome of the thus far best-characterized myxobacterium, Myxococcus xanthus, in one step. The successful integration and expression of the Myxothiazol biosynthetic genes in M. xanthus results in the production of Myxothiazol in yields comparable to the natural producer strain.
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Rational Design of a Bimodular Model System for the Investigation of Heterocyclization in Nonribosomal Peptide Biosynthesis
Chemistry & biology, 2004Co-Authors: Thomas Duerfahrt, Rolf Müller, Katrin Eppelmann, Mohamed A. MarahielAbstract:Cyclization (Cy) domains in NRPS catalyze the heterocyclization of cysteine and serine/threonine to thiazoline and oxazoline rings. A model system consisting of the first two modules of bacitracin synthetase A fused to the thioesterase (Te) domain of tyrocidine synthetase was constructed (BacA1-2-Te) and shown to be active in production of the heterocyclic IleCys(thiazoline). Based on this model system, the feasibility of Cy domain module fusions was investigated by replacing the BacA2 Cy-A-PCP-module with modules of MbtB and MtaD from the biosynthesis systems of mycobactin and Myxothiazol, revealing the formation of novel heterocyclic dipeptides. To dissect the reaction sequence of the Cy domain in peptide bond formation and heterocyclization, several residues of the BacA1-2-Te Cy domain were analyzed by mutagenesis. Two mutants exhibited formation of the noncyclic dipeptide, providing clear evidence for the independence of condensation and cyclization.
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Markerless Mutations in the Myxothiazol Biosynthetic Gene Cluster: A Delicate Megasynthetase with a Superfluous Nonribosomal Peptide Synthetase Domain
Chemistry & Biology, 2003Co-Authors: Stefan Weinig, Taifo Mahmud, Rolf MüllerAbstract:Myxobacteria are efficient producers of natural products with various biological activities. Nevertheless, genetic systems for markerless mutagenesis are only available for Myxococcus xanthus DK1622, a strain that is not known to produce any secondary metabolites. We report here the development of such a technique for Stigmatella aurantiaca DW4/3-1, a multiproducer of natural products. The system is used to further characterize the combined polyketide synthase/peptide synthetase system responsible for Myxothiazol biosynthesis. Analysis of a series of point mutations and in-frame deletions shows that the Myxothiazol megasynthetase is a rather specialized and delicate system that is hardly capable of processing unnatural intermediates. An in-frame deletion of the MtaC oxidation domain results in the production of unchanged bis-thiazole Myxothiazol instead of the expected thiazoline-thiazole derivative of the compound. This shows that this domain within a nonribosomal peptide synthetase is not necessary for product formation.
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Melithiazol Biosynthesis: Further Insights into Myxobacterial PKS/NRPS Systems and Evidence for a New Subclass of Methyl Transferases
Chemistry & Biology, 2003Co-Authors: Stefan Weinig, Taifo Mahmud, Hans-jürgen Hecht, Rolf MüllerAbstract:Abstract A DNA region of 41.847 base pairs from Melittangium lichenicola Me l46 is shown to be responsible for the biosynthesis of the potent electron transport inhibitor melithiazol. Melithiazol is formed by a combined polyketide synthase/peptide synthetase system resembling the Myxothiazol megasynthetase from Stigmatella aurantiaca DW4/3-1. Both natural products share an almost identical core region but employ different starter molecules. Additionally, melithiazol contains a terminal methyl ester instead of the amide moiety found in Myxothiazol. Similar to Myxothiazol formation, the methyl ester is formed via an amide intermediate, which is converted by a hydrolase and an unusual type of SAM (S-adenosyl-L-methionine)-dependent methyltransferase into the methyl ester. When transferred into the Myxothiazol A (amide) producer, these two genes lead to the formation of the methyl ester of Myxothiazol. The methyl transferase described is a member of a protein subfamily of a previously unknown function lacking a typical SAM binding motif.
Junying Yuan - One of the best experts on this subject based on the ideXlab platform.
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mitochondrial electron transport chain complex iii is required for antimycin a to inhibit autophagy
Chemistry & Biology, 2011Co-Authors: Mingzhi Jin, Yu Cai, Hongguang Xia, Kai Long, Junli Liu, Junying YuanAbstract:Autophagy is a cellular lysosome-dependent catabolic mechanism mediating the turnover of intracellular organelles and long-lived proteins. We show that antimycin A, a known inhibitor of mETC complex III, can inhibit autophagy. A structural and functional study shows that four close analogs of antimycin A that have no effect on mitochondria inhibition also do not inhibit autophagy, whereas Myxothiazol, another mETC complex III inhibitor with unrelated structure to antimycin A, inhibits autophagy. Additionally, antimycin A and Myxothiazol cannot inhibit autophagy in mtDNA-depleted H4 and mtDNA-depleted HeLa cells. These data suggest that antimycin A inhibits autophagy through its inhibitory activity on mETC complex III. Our data suggest that mETC complex III may have a role in mediating autophagy induction.
Taketoshi Uzawa - One of the best experts on this subject based on the ideXlab platform.
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The interaction of the Rieske iron-sulfur protein with occupants of the Qo-site of the bc1 complex, probed by electron spin echo envelope modulation
Archaea, 2015Co-Authors: Taketoshi Uzawa, Toshio Iwasaki, Sergei A. DikanovAbstract:The bifurcated reaction at the Qo-site of the bc1 com-plex provides the mechanistic basis of the proton pump-ing activity through which the complex conserves redox energy in the proton gradient. Structural information about the binding of quinone at the site is lacking, be-cause the site is vacant in crystals of the native com-plexes. We now report the first structural characteriza-tion of the interaction of the native quinone occupant with the Rieske iron-sulfur protein in the bc1 complex of Rhodobacter sphaeroides, using high resolution EPR. We have compared the binding configuration in the presence of quinone with the known structures for the complex with stigmatellin and Myxothiazol. We have shown by using EPR and orientation-selective electron spin echo envelope modulation (ESEEM) measurement
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The interaction of the Rieske iron-sulfur protein with occupants of the Qo-site of the bc1 complex, probed by electron spin echo envelope modulation.
The Journal of biological chemistry, 2001Co-Authors: Rimma I. Samoilova, Antony R Crofts, Derrick R. J. Kolling, Taketoshi Uzawa, Toshio Iwasaki, Sergei A. DikanovAbstract:The bifurcated reaction at the Q(o)-site of the bc(1) complex provides the mechanistic basis of the proton pumping activity through which the complex conserves redox energy in the proton gradient. Structural information about the binding of quinone at the site is lacking, because the site is vacant in crystals of the native complexes. We now report the first structural characterization of the interaction of the native quinone occupant with the Rieske iron-sulfur protein in the bc(1) complex of Rhodobacter sphaeroides, using high resolution EPR. We have compared the binding configuration in the presence of quinone with the known structures for the complex with stigmatellin and Myxothiazol. We have shown by using EPR and orientation-selective electron spin echo envelope modulation (ESEEM) measurements of the iron-sulfur protein that when quinone is present in the site, the isotropic hyperfine constant of one of the N(delta) atoms of a liganding histidine of the [2Fe-2S] cluster is similar to that observed when stigmatellin is present and different from the configuration in the presence of Myxothiazol. The spectra also show complementary differences in nitrogen quadrupole splittings in some orientations. We suggest that the EPR characteristics, the ESEEM spectra, and the hyperfine couplings reflect a similar interaction between the iron-sulfur protein and the quinone or stigmatellin and that the N(delta) involved is that of a histidine (equivalent to His-161 in the chicken mitochondrial complex) that forms both a ligand to the cluster and a hydrogen bond with a carbonyl oxygen atom of the Q(o)-site occupant.
