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Toivo Kallas - One of the best experts on this subject based on the ideXlab platform.

  • Modification of inhibitor binding sites in the cytochrome bf complex by directed mutagenesis of cytochrome b(6) in Synechococcus sp. PCC 7002.
    Biochimica et biophysica acta, 2001
    Co-Authors: T Lee, Sabine U Metzger, Yoon Shin Cho, John Whitmarsh, Toivo Kallas
    Abstract:

    The cytochrome bf complex, which links electron transfer from photosystem II to photosystem I in oxygenic photosynthesis, has not been amenable to site-directed mutagenesis in cyanobacteria. Using the cyanobacterium Synechococcus sp. PCC 7002, we have successfully modified the cytochrome b(6) subunit of the cytochrome bf complex. Single amino acid substitutions in cytochrome b(6) at the positions D148, A154, and S159 revealed altered binding of the quinol-oxidation inhibitors 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB), myxothiazol, and Stigmatellin. Cytochrome bf and mitochondrial-type cytochrome bc(1) complexes are closely related in structure and function but exhibit quite different inhibitor specificities. Cytochrome bf complexes are insensitive to myxothiazol and sensitive to DBMIB, whereas cytochrome bc(1) complexes are sensitive to myxothiazol and relatively insensitive to DBMIB. Measurements of flash-induced and steady-state electron transfer rates through the cytochrome bf complex revealed increased resistance to DBMIB in the mutants A154G and S159A, increased resistance to Stigmatellin in A154G, and created sensitivity to myxothiazol in the mutant D148G. Therefore these mutations made the cytochrome bf complex more like the cytochrome bc(1) complex. This work demonstrates that cyanobacteria can be used as effective models to investigate structure-function relationships in the cytochrome bf complex.

  • Modification of inhibitor binding sites in the cytochrome bf complex by directed mutagenesis of cytochrome b6 in Synechococcus sp. PCC 7002
    Biochimica et Biophysica Acta (BBA) - Bioenergetics, 2001
    Co-Authors: Tou-xang Lee, Sabine U Metzger, Yoon Shin Cho, John Whitmarsh, Toivo Kallas
    Abstract:

    AbstractThe cytochrome bf complex, which links electron transfer from photosystem II to photosystem I in oxygenic photosynthesis, has not been amenable to site-directed mutagenesis in cyanobacteria. Using the cyanobacterium Synechococcus sp. PCC 7002, we have successfully modified the cytochrome b6 subunit of the cytochrome bf complex. Single amino acid substitutions in cytochrome b6 at the positions D148, A154, and S159 revealed altered binding of the quinol-oxidation inhibitors 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB), myxothiazol, and Stigmatellin. Cytochrome bf and mitochondrial-type cytochrome bc1 complexes are closely related in structure and function but exhibit quite different inhibitor specificities. Cytochrome bf complexes are insensitive to myxothiazol and sensitive to DBMIB, whereas cytochrome bc1 complexes are sensitive to myxothiazol and relatively insensitive to DBMIB. Measurements of flash-induced and steady-state electron transfer rates through the cytochrome bf complex revealed increased resistance to DBMIB in the mutants A154G and S159A, increased resistance to Stigmatellin in A154G, and created sensitivity to myxothiazol in the mutant D148G. Therefore these mutations made the cytochrome bf complex more like the cytochrome bc1 complex. This work demonstrates that cyanobacteria can be used as effective models to investigate structure–function relationships in the cytochrome bf complex

Bernard L. Trumpower - One of the best experts on this subject based on the ideXlab platform.

  • 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, 2007
    Co-Authors: Raul Covian, Thomas Kleinschroth, Bernd Ludwig, Bernard L. Trumpower
    Abstract:

    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.

  • A comparison of Stigmatellin conformations, free and bound to the photosynthetic reaction center and the cytochrome bc1 complex.
    Journal of molecular biology, 2007
    Co-Authors: C. Roy D. Lancaster, Carola Hunte, Jack Kelley, Bernard L. Trumpower, Robert Ditchfield
    Abstract:

    We describe in detail the conformations of the inhibitor Stigmatellin in its free form and bound to the ubiquinone-reducing (QB) site of the reaction center and to the ubiquinol-oxidizing (Qo) site of the cytochrome bc1 complex. We present here the first structures of a stereochemically correct Stigmatellin in complexes with a bacterial reaction center and the yeast cytochrome bc1 complex. The conformations of the inhibitor bound to the two enzymes are not the same. We focus on the orientations of the Stigmatellin side-chain relative to the chromone head group, and on the interaction of the Stigmatellin side-chain with these membrane protein complexes. The different conformations of Stigmatellin found illustrate the structural variability of the Q sites, which are affected by the same inhibitor. The free rotation about the χ1 dihedral angle is an essential factor for allowing Stigmatellin to bind in both the reaction center and the cytochrome bc1 pocket.

  • regulatory interactions between ubiquinol oxidation and ubiquinone reduction sites in the dimeric cytochrome bc1 complex
    Journal of Biological Chemistry, 2006
    Co-Authors: Raul Covian, Bernard L. Trumpower
    Abstract:

    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.

  • The interaction of atovaquone with the P. carinii cytochrome bc1 complex.
    Journal of Eukaryotic Microbiology, 2001
    Co-Authors: Steven R. Meshnick, Edward A. Berry, Jurcen B Nett, Powel Kazanjian, Bernard L. Trumpower
    Abstract:

    Author(s): Trumpower, B. | Abstract: Atovaquone is an important second-line therapeutic and prophylactic agent for Pneumocystis carinii pneumonia although it was originally developed as an antimalarial [11]. For malaria, atovaquone is now only used in combination with proguanil, another antimalarial. This combination (Malaronel) is used because atovaquone monotherapy leads to resistance [9]. Atovaquone is structurally similar to ubiquinone (CoQ). UHDBT and Stigmatellin are other CoQ analogs that have been well studied in experimental systems. Both UHDBT and Stigmatellin are known to inhibit electron transport by binding to the Q0 site of the cytochrome bc1 complex. Atovaquone probably acts at the same site, since it has also been shown to inhibit electron transport in both malaria and P. carinii [4, 6]. This mechanism could also explain the rapid emergence of resistance to monotherapy; mutations could arise easily in cytochrome b, since it is encoded in the mitochondrial genome where the spontaneous mutation rate is 10x higher than in nucleus [1].

  • Determination of the binding rate constants of Stigmatellin and UHDBT to bovine cytochrome bc1 complex by cytochrome c1 oxidation
    FEBS letters, 1999
    Co-Authors: Li Zhang, C Snyder, Bernard L. Trumpower
    Abstract:

    Based on the high electron transfer rate between the [2Fe-2S] cluster and heme c(1) and the elevation of the redox midpoint potential of iron sulfur protein (ISP) upon binding of certain Qo inhibitors, the binding rate constants of Stigmatellin and UHDBT to the cytochrome bc(1) complex were determined using a stopped-flow rapid scanning technique. Assuming that the intramolecular electron transfer from ISP to cytochrome c(1) is much faster than the binding of inhibitors, the rate of the inhibitor binding can be determined by the rate of cytochrome c(1) oxidation. The binding rate constants were calculated to be 1.0x10(5) and 2.3x10(5) M(-1) s(-1) at pH 7.5 for Stigmatellin and UHDBT, respectively. The binding rate constant of UHDBT is pH dependent and that of Stigmatellin is not.

Daniel Uguen - One of the best experts on this subject based on the ideXlab platform.

Natesan Sivakumar - One of the best experts on this subject based on the ideXlab platform.

  • Stigmatellin y an anti biofilm compound from bacillus subtilis br4 possibly interferes in pqs pqsr mediated quorum sensing system in pseudomonas aeruginosa
    Bioorganic & Medicinal Chemistry Letters, 2017
    Co-Authors: Seenivasan Boopathi, Rajesh Vashisth, Prabu Manoharan, Ruckmani Kandasamy, Natesan Sivakumar
    Abstract:

    Hitherto this is the first report pertaining to production of biofilm inhibitory compound(s) (BIC) from Bacillus subtilis BR4 against Pseudomonas aeruginosa (ATCC 27853) coupled with production optimization. In order to achieve this, combinations of media components were formulated by employing statistical tools such as Plackett-Burman analysis and central composite rotatable design (CCRD). It was evident that at 35mlL-1 glycerol and 3.8gL-1 casamino acid, anti-biofilm activity and production of extracellular protein significantly increased by 1.5-fold and 1.2-fold, respectively. These results corroborate that the combination of glycerol and casamino acid plays a key role in the production of BIC. Further, metabolic profiling of BIC was carried out using liquid chromatography/tandem mass spectrometry (LC-MS/MS) based on m/z value. The presence of Stigmatellin Y was predicted with monoisotopic neutral mass of 484.2825Da. In support of optimization study, higher production of BIC was confirmed in the optimized-media-grown BR4 (OPT-BR4) than in the ideal-media-grown BR4 (ID-BR4) by LC-MS/MS analysis. PqsR in P. aeruginosa is a potential target for anti-virulent therapy. Molecular docking study has revealed that Stigmatellin Y interacts with PqsR in the similar orientation like a cognate signal (PQS) and synthetic inhibitor. In addition, Stigmatellin Y was found to exhibit interaction with four more amino acid residues of PqsR to establish strong affinity. Stigmatellin Y thus might play a role of competitor for PQS to distract PQS-PqsR mediated communication in P. aeruginosa. The present investigation thus paves new avenues to develop anti-Pseudomonas virulent therapy.

  • Stigmatellin Y – An anti-biofilm compound from Bacillus subtilis BR4 possibly interferes in PQS–PqsR mediated quorum sensing system in Pseudomonas aeruginosa
    Bioorganic & medicinal chemistry letters, 2017
    Co-Authors: Seenivasan Boopathi, Rajesh Vashisth, Prabu Manoharan, Ruckmani Kandasamy, Natesan Sivakumar
    Abstract:

    Hitherto this is the first report pertaining to production of biofilm inhibitory compound(s) (BIC) from Bacillus subtilis BR4 against Pseudomonas aeruginosa (ATCC 27853) coupled with production optimization. In order to achieve this, combinations of media components were formulated by employing statistical tools such as Plackett-Burman analysis and central composite rotatable design (CCRD). It was evident that at 35mlL-1 glycerol and 3.8gL-1 casamino acid, anti-biofilm activity and production of extracellular protein significantly increased by 1.5-fold and 1.2-fold, respectively. These results corroborate that the combination of glycerol and casamino acid plays a key role in the production of BIC. Further, metabolic profiling of BIC was carried out using liquid chromatography/tandem mass spectrometry (LC-MS/MS) based on m/z value. The presence of Stigmatellin Y was predicted with monoisotopic neutral mass of 484.2825Da. In support of optimization study, higher production of BIC was confirmed in the optimized-media-grown BR4 (OPT-BR4) than in the ideal-media-grown BR4 (ID-BR4) by LC-MS/MS analysis. PqsR in P. aeruginosa is a potential target for anti-virulent therapy. Molecular docking study has revealed that Stigmatellin Y interacts with PqsR in the similar orientation like a cognate signal (PQS) and synthetic inhibitor. In addition, Stigmatellin Y was found to exhibit interaction with four more amino acid residues of PqsR to establish strong affinity. Stigmatellin Y thus might play a role of competitor for PQS to distract PQS-PqsR mediated communication in P. aeruginosa. The present investigation thus paves new avenues to develop anti-Pseudomonas virulent therapy.

Fevzi Daldal - One of the best experts on this subject based on the ideXlab platform.

  • Role of the Neck Region of the Rieske Fe-S Protein in Q o Site Catalysis
    Photosynthesis: Mechanisms and Effects, 1998
    Co-Authors: Elisabeth Darrouzet, Maria Valkova-valchanova, Fevzi Daldal
    Abstract:

    Ubiquinol:cytochrome c oxidoreductase, or bc1 complex, plays a central role in both respiratory and photosynthetic processes. While the mitochondrial enzyme is made up of eleven subunits, numerous bacteria harbor a simpler homologous complex which contains only three subunits forming the core portion of the mitochondrial enzyme. These subunits bear two b-hemes (bL and bH) one c-heme (c1) and one I2Fe-25 I cluster as cofactors, and form two active domains called QO and Qi sites (for review see 1–2). In bacteria, the genes encoding for these three core proteins are clustered in an operon called pet or flic. In recent years, several structures for mitochondrial bc1 complexes have been published (3–5) but none of them fit completely with the entire catalytic mechanism. The distance between the iron sulfur cluster and heme c1 estimated from the structure of Xia et al. (31 A) cannot support fast electron transfer kinetics between these two redox centers. On the other hand, the cleft observed between the Rieske subunit and b subunit in the case of Zhang et al. structure is hardly compatible with the fact that some inhibitors of the Qo site and quinol and quinone interact with both the Rieske subunit and the cyt b subunit. Along the years the idea that the Rieske subunit may move to accommodate all the experimental data has emerged. In particular the recent crystallographic data obtained by Zhang and colleagues in presence and absence of Stigmatellin revealed a movement of the Rieske head of about 16 A with a rotation of 57° (4). During this movement, the hydrophobic membrane anchor remains at the same position so that the binding of Stigmatellin seems to require an extension of the connecting domain (the neck) between the tail and the head of the Rieske subunit. But whether or not this movement, and in particular the flexibility of the Rieske neck, is really required for the activity of the bc1 complex remains unknown. Using Rhodobacter capsulatus bc1 complex as a model we were first able to show biochemically that the neck region undergoes changes when Stigmatellin binds, and that a full length Rieske with its amino terminal part is required for the activity of the complex. Later on, mutants engineered genetically in this region revealed that the length and flexibility and not the precise nature of each amino acid of this loop is important for the stability and activity of the complex.

  • The amino-terminal portion of the Rieske iron-sulfur protein contributes to the ubihydroquinone oxidation site catalysis of the Rhodobacter capsulatus bc1 complex.
    Biochemistry, 1997
    Co-Authors: Gaël Brasseur, Tomoko Ohnishi, Vladimir D. Sled, Ursula Liebl, Fevzi Daldal
    Abstract:

    The Rieske iron-sulfur (Fe-S) protein subunit of bc1 complexes contains in its carboxyl-terminal part two highly conserved hexapeptide motifs (box I and box II) that include the four amino acid ligands of its [2Fe-2S] cluster. In the preceding paper [Liebl, U., Sled, V., Brasseur, G., Ohnishi, T., & Daldal, F. (1997) Biochemistry 36, 11675-11684], the effects of mutations at two of the nonliganding residues [threonine (T) 134 and leucine (L) 136 in the Rhodobactercapsulatus Rieske Fe-S protein] of box I have been described. In this work, interactions between the occupants of the Qo site of the bc1 complex (UQ/UQH2 and the inhibitors Stigmatellin and myxothiazol) and the [2Fe-2S] cluster of the Rieske Fe-S protein were probed by isolating photosynthesis-proficient (Ps+) revertants of the Ps- mutants L136R, -H, -D and -G. These revertants contained either a single substitution at the original position 136 or an additional mutation located in the amino-terminal part of the Fe-S protein at either position 44 or 46. The same-site revertants L136A and -Y grew well under photosynthetic conditions and contained highly active bc1 complexes but exhibited modified EPR spectra both in the presence and in the absence of Stigmatellin. Unexpectedly, they were highly resistant to Stigmatellin (StiR) and hypersensitive to myxothiazol (MyxHS) in vivo, demonstrating for the first time that mutations located in the Fe-S subunit confer resistance to Stigmatellin. The [2Fe-2S] cluster of the same-site revertants responded weakly to the Qpool redox state and had redox midpoint potential (Em7) values (around 265 mV) lower than those of their wild type counterpart (about 310 mV). On the other hand, the second-site revertants L136H/V44L, L136G/V44F, and L136G/A46T, -V, or -P supported photosynthetic growth poorly, were StiR and MyxHS, and contained barely active bc1 complexes. Like the same-site revertants, they exhibited modified EPR spectra both in the presence and in the absence of Stigmatellin and had perturbed Qo site occupancy. In addition, they contained substoichiometric amounts of the Fe-S protein with respect to the other subunits of the bc1 complex. The Em7 values of the [2Fe-2S] cluster of these double mutants were lower (around 245 mV) than that of the wild type strain but appreciably higher than those of their Ps- parents (about 200 mV for L136G). In order to define the molecular nature of the suppression mediated by the second-site mutations, the single mutants V44L and -F and A46T and -V were constructed in the absence of the original mutations at position 136. These mutants behaved like a wild type strain with respect to their Ps+ growth ability, inhibitor sensitivity, EPR spectra of their [2Fe-2S] cluster, and response to Stigmatellin or to the Qpool redox state. But surprisingly, the Em7 values of their [2Fe-2S] cluster were much higher (about 385 mV) than that of a wild type strain. These findings demonstrated for the first time that the amino-terminal part of the Rieske Fe-S protein encompassing residues 44 and 46 is important not only for the structure and function of the Qo site of the bc1 complex but also for the properties of its [2Fe-2S] cluster.

  • Conserved nonliganding residues of the Rhodobacter capsulatus Rieske iron-sulfur protein of the bc1 complex are essential for protein structure, properties of the [2Fe-2S] cluster, and communication with the quinone pool.
    Biochemistry, 1997
    Co-Authors: Ursula Liebl, Tomoko Ohnishi, Gaël Brasseur, Vladimir D. Sled, Fevzi Daldal
    Abstract:

    The iron-sulfur (Fe-S) protein subunit of the bc1 complex, known as the Rieske protein, contains a high-potential [2Fe-2S] cluster ligated by two nitrogen and two sulfur atoms to its apoprotein. Earlier work indicated that in Rhodobacter capsulatus these atoms are provided by two cysteine (C133 and C153) and two histidine (H135 and H156) residues, located at the carboxyl-terminal end of the protein [Davidson, E., Ohnishi, T., Atta-Asafo-Adjei, E., & Daldal, F. (1992) Biochemistry 31, 3342-3351]. These ligands are part of the conserved sequences C133THLGC138 (box I) and C153PCHGS158 (box II) and affect the properties of the Fe-S protein and its [2Fe-2S] cluster. In this work, the role of amino acid side chains at positions 134 and 136, adjacent to the cluster ligands in box I, was probed by using site-directed mutagenesis and biophysical analyses. These positions were substituted with R, D, H, and G to probe the effect of charged, polar, large, and small amino acid side chains on the properties of the [2Fe-2S] cluster. Of the mutants obtained T134R, -H, and -G were photosynthetically competent (Ps+) but contained Fe-S proteins with redox midpoint potentials (Em7) 50-100 mV lower than that of a wild type strain. In contrast, T134D was Ps- and contained no detectable [2Fe-2S] cluster, although it reverted frequently to Ps+ by substitution of D with N. On the other hand, all L136 mutants were Ps-, the EPR characteristics of their [2Fe-2S] cluster were perturbed, and they were unable to sense the Qpool redox state or to bind Stigmatellin properly. The overall data indicated that replacement of the amino acid side chain at position 134 of the Fe-S protein affects mainly the Em7 and oxygen sensitivity of the [2Fe-2S] cluster without abolishing its function, while substitutions at position 136 perturb drastically its ability to monitor the Qpool redox state and its interaction with the Qo site inhibitor Stigmatellin. These two distinct phenotypes of box I T134 and L136 mutants are discussed with regard to the recently published three-dimensional structure of the water soluble part of the bovine heart mitochondrial Rieske Fe-S protein.

  • Roles in inhibitor recognition and quinol oxidation of the amino acid side chains at positions of cytb providing resistance to Qo‐inhibitors of the bc1 complex from Rhodobacter capsulatus
    Molecular microbiology, 1993
    Co-Authors: Mariko Tokito, Fevzi Daldal
    Abstract:

    The substitutions M1401, F144S and L, G152S, T163A and V333A in cytochrome b of the ubiquinol-cytochrome c oxidoreductase (bc1 complex) from Rhodobacter capsulatus provide resistance to the quinol oxidation (Qo) inhibitors myxothiazol, mucidin and Stigmatellin. Site-directed mutagenesis with degenerate primers was used to define the role of these positions in inhibitor recognition and quinol oxidation, and a collection of various substitutions at each of these positions was obtained. The effects of these mutations on quinol oxidation, nature and level of inhibitor resistance, prosthetic group incorporation and assembly of the complex were analysed. Most of these mutations, unlike those at position 158 reported earlier, yielded functional bc1 complexes able to support the photosynthetic growth of R. capsulatus. However, they perturbed steady-state quinol oxidation and inhibitor recognition indicating that they are important for the function of the Qo site. In particular, the presence of a methyl group on the beta-carbon (Ile and Val residues) at position 140, the absence of an aromatic ring (Phe, Tyr and Trp residues) at position 144 and the loss of residues with small side chains (Gly and Ala) at position 152 correlated with resistance to myxothiazol. On the other hand, no myxothiazol resistance was observed with the substitutions at positions 163 and 333 suggesting that they affected solely the recognition of Stigmatellin. Five substitutions, M140R, F144H and R, G152P and T163R, yielded photosynthesis-deficient mutants with assembled but impaired bc1 complexes. Unexpectedly, two substitutions at position 163 (T to F or P) yielded mutants lacking the three subunits of the bc1 complex indicating that this position affects its assembly or stability in vivo. These findings are discussed in terms of the contributions of these residues to inhibitor recognition and quinol oxidation at the Qo site of the bc1 complex.