Christophe Bruel

Massachusetts Institute of Technology, Cambridge, Massachusetts, United States

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Publications (8)29.76 Total impact

  • Yudong Wang · Christophe Bruel · Le Yan · Diana S. Beattie
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    ABSTRACT: Previously, we reported that the carboxyl-reacting reagent DCCD, and its fluorescent derivative NCD-4 binds covalently to aspartate-160 localized in amphipathic helix cd of the CD loop connecting membrane-spanning helices C and D of cytochrome b (Wang et al., 1995). We have investigated the fluorescent properties of NCD-4 to probe possible changes in the cd helix resulting from the binding of exogenous ubiquinol analogues to the bc1 complex. Preincubation of the bc1 complex with the reduced substrate analogues, DQH2, DBH2, and Q6H2 resulted in 20-40% increase in the fluorescence emission intensity of NCD-4 and a 10-20% increase in the binding of [14C]DCCD to the bc1 complex. By contrast, preincubation with the oxidized analogues DQ. DB, and Q6 resulted in a 20-40% decrease in the fluorescence emission intensity of NCD-4 and a 20-40% decrease in the binding of [14C]DCCD to the bc1 complex. Moreover, addition of the reduced ubiquinols to the bc1 complex preincubated with NCD-4 resulted in a blue shift in the fluorescence emission spectrum. In addition, incubation of the cytochrome bc1 complex reconstituted into proteoliposomes with both reduced and oxidized ubiquinol analogues resulted in changes in the quenching of NCD-4 fluorescence by CAT-16, the spin-label probe that intercalates at the membrane surface. These results indicate that the addition of exogenous ubiquinol to the bc1 complex may result in changes in the cd helix leading to a more hydrophobic environment surrounding the NCD-4 binding site. By contrast, preincubation with the inhibitors of electron transfer through the bc1 complex had no effect on the binding of NCD-4 to the bc1 complex or on the fluorescent emission spectra, which suggests that the binding of the inhibitors does not result in changes in the environment of the NCD-4 binding site.
    No preview · Article · Nov 1998 · Journal of Bioenergetics
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    ABSTRACT: A cDNA carrying the Rip1 gene, which encodes the Rieske iron-sulfur protein of Schizosaccharomyces pombe, has been cloned by complementing the respiratory deficiency of a Saccharomyces cerevisiae strain in which the endogenous copy of the RIP1 gene has been deleted. The deduced amino acid sequences of the S. pombe and S. cerevisiae iron-sulfur proteins are 50% identical, with the highest region of identity being in the C termini of the proteins, where the 2Fe:2S cluster is bound. When expressed in the S. cerevisiae deletion strain, the S. pombe iron-sulfur protein restores 25–30% of the ubiquinol-cytochrome c reductase activity. The kinetics of cytochrome c reduction, the effects of inhibitors which act at defined sites in the cytochrome bc1 complex, and the optical properties of cytochrome b in membranes from the S. cerevisiae deletion strain complemented with S. pombe iron-sulfur protein indicate that the S. pombe protein interacts with cytochrome b to restore an apparently normal ubiquinol oxidase site, but that interaction between the iron-sulfur protein and cytochrome c1 is partially impaired. This is the first heterologous replacement of an electron transfer protein in a respiratory enzyme complex in S. cerevisiae.
    Full-text · Article · Jul 1996 · Journal of Biological Chemistry
  • Christophe Bruel · Robert Brasseur · Bernard L. Trumpower
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    ABSTRACT: We have investigated the function of subunit 8 of the cytochromebc 1 complex by generating six site-directed mutants, F46C, R51S, P62V, G64A, R91N, and W69-stop, in the clonedQCR8 gene and expressing the mutated genes in aSaccharomyces cerevisiae strain in which the chromosomal copy ofQCR8 is deleted. The W69-stop mutation impairs assembly of thebc 1 complex and growth of yeast on nonfermentable carbon sources as does deletion ofQCR8 [Maarse, A. C., De Haan, M., Schoppink, P. J., Berden, J. A., and Grivell, L. A. (1988).Eur. J. Biochem. 172, 179–184], implying that the C-terminus of subunit 8 is important for assembly and/or the stability of thebc 1 complex. The F46C, R51S, P62V, G64A, and R91N mutations do not affect the growth of yeast on nonfermentable carbon sources, not do they lower the activity or alter the inhibitor sensitivity of thebc 1 complex. Rather, some of the mutations increase the cytochromec reductase activity of thebc 1 complex by as much as 40%. However, succinate-ubiquinone reductase activity was consistently reduced 40–60% in mitochondrial membranes from these mutants, while NADH-ubiquinone reductase activity was not affected. In addition, the activation of succinate-ubiquinone reductase activity by succinate was diminished by the F46C, R51S, P62V, and G64A mutations. These results indicate that the cytochromebc 1 complex participates in electron transfer from succinate to ubiquinonein situ and also suggest an interaction between succinate-ubiquinone reductase and cytochromebc 1 complex which involves subunit 8 of thebc 1 complex.
    No preview · Article · Mar 1996 · Journal of Bioenergetics
  • Christophe Bruel · Robert Brasseur · Bernard L. Trumpower
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    ABSTRACT: We have investigated the function of subunit 8 of the cytochromebc 1 complex by generating six site-directed mutants, F46C, R51S, P62V, G64A, R91N, and W69-stop, in the clonedQCR8 gene and expressing the mutated genes in aSaccharomyces cerevisiae strain in which the chromosomal copy ofQCR8 is deleted. The W69-stop mutation impairs assembly of thebc 1 complex and growth of yeast on nonfermentable carbon sources as does deletion ofQCR8 [Maarse, A. C., De Haan, M., Schoppink, P. J., Berden, J. A., and Grivell, L. A. (1988).Eur. J. Biochem. 172, 179–184], implying that the C-terminus of subunit 8 is important for assembly and/or the stability of thebc 1 complex. The F46C, R51S, P62V, G64A, and R91N mutations do not affect the growth of yeast on nonfermentable carbon sources, not do they lower the activity or alter the inhibitor sensitivity of thebc 1 complex. Rather, some of the mutations increase the cytochromec reductase activity of thebc 1 complex by as much as 40%. However, succinate-ubiquinone reductase activity was consistently reduced 40–60% in mitochondrial membranes from these mutants, while NADH-ubiquinone reductase activity was not affected. In addition, the activation of succinate-ubiquinone reductase activity by succinate was diminished by the F46C, R51S, P62V, and G64A mutations. These results indicate that the cytochromebc 1 complex participates in electron transfer from succinate to ubiquinonein situ and also suggest an interaction between succinate-ubiquinone reductase and cytochromebc 1 complex which involves subunit 8 of thebc 1 complex.
    No preview · Article · Feb 1996 · Journal of Bioenergetics
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    ABSTRACT: Four mutations in the mitochondrial cytochromeb ofS. cerevisiae have been characterized with respect to growth capacities, catalytic properties, ATP/2e − ratio, and transmembrane potential. The respiratory-deficient mutant G137E and the three pseudo-wild type revertants E137 + I147F, E137 + C133S, and E137 + N256K were described previously (Tron and Lemesle-Meunier, 1990; Di Ragoet al., 1990a). The mutant G137E is unable to grow on respiratory substrates but its electron transfer activity is partly conserved and totally inhibited by antimycin A. The secondary mutations restore the respiratory growth at variable degree, with a phosphorylation efficiency of 12–42% as regards the parental wild type strain, and result in a slight increase in the various electron transfer activities at the level of the whole respiratory chain. The catalytic efficiency for ubiquinol was slightly (G137E) or not affected (E137 + I147F, E137 + C133S, and E137 + N256K) in these mutants. Mutation G137E induces a decrease in the ATP/2e − ratio (50% of the W.T. value) and transmembrane potential (60% of the W.T. value) at thebc1 level, whereas the energetic capacity of the cytochrome oxidase is conserved. Secondary mutations I147F, C133S, and N256K partly restore the ATP/ 2e − ratio and the transmembrane potential at thebc1 complex level. The results suggest that a partial decoupling of thebc1 complex is induced by the cytochromeb point mutation G137E. In the framework of the protonmotive Q cycle, this decoupling can be explained by the existence of a proton wire connecting centers P and N in the wild typebc1 complex which may be amplified or uncovered by the G137E mutation when the bc1 complex is functioning.
    No preview · Article · Nov 1995 · Journal of Bioenergetics
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    ABSTRACT: Trp-142 is a highly conserved residue of the cytochrome b subunit in the bc1 complexes. To study the importance of this residue in the quinol oxidation catalyzed by the bc1 complex, we characterized four yeast mutants with arginine, lysine, threonine, and serine at position 142. The mutant W142R was isolated previously as a respiration-deficient mutant unable to grow on non-fermentable carbon sources (Lemesle-Meunier, D., Brivet-Chevillotte, P., di Rago, J.-P, Slonimski, P.P., Bruel, C., Tron, T., and Forget, N. (1993) J. Biol. Chem. 268, 15626-15632). The mutants W142K, W142T, and W142S were obtained here as respiration-sufficient revertants from mutant W142R. Mutant W142R exhibited a decreased complex II turnover both in the presence and absence of antimycin A; this suggests that the structural effect of W142R in the bc1 complex probably interferes with the correct assembly of the succinate-ubiquinone reductase complex. The mutations resulted in a parallel decrease in turnover number and apparent Km, with the result that there was no significant change in the second-order rate constant for ubiquinol oxidation. Mutants W142K and W142T exhibited some resistance toward myxothiazol, whereas mutant W142R showed increased sensitivity. The cytochrome cc1 reduction kinetics were found to be severely affected in mutants W142R, W142K, and W142T. The respiratory activities and the amounts of reduced cytochrome b measured during steady state suggest that the W142S mutation also modified the quinol-cytochrome c1 electron transfer pathway. The cytochrome b reduction kinetics through center P were affected when Trp-142 was replaced with arginine or lysine, but not when it was replaced with threonine or serine. Of the four amino acids tested at position 142, only arginine resulted in a decrease in cytochrome b reduction through center N. These findings are discussed in terms of the structure and function of the quinol oxidation site and seem to indicate that Trp-142 is not critical to the kinetic interaction of ubiquinol with the reductase, but plays an important role in the electron transfer reactions that intervene between ubiquinol oxidation and cytochrome c1 reduction.
    Preview · Article · Oct 1995 · Journal of Biological Chemistry
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    CHRISTOPHE BRUEL · DANIELLE LEMESLE-MEUNIER

    Preview · Article · Mar 1994 · Biochemical Society Transactions
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    ABSTRACT: We characterized six novel missense mutations in mitochondrial cytochrome b (C133Y, W142R, S206L, M221K, L282F, and G340E) which impair the respiratory growth of yeast and which have differential effects on the functioning and assembly of the bc1 complex. The mutations have been mapped genetically in exons of the mitochondrial gene coding for apocytochrome b and their nucleotide sequence established. The mutants help to better define the topographical and primary sequence location of the ubiquinol oxidase (center P) and ubiquinone reductase (center N) sites on cytochrome b. Two mutants (C133Y and S206L) resulted in an active assembled complex, with selective disturbances of heme 565 and heme 562, respectively, which is consistent with the assignment of the axial ligands of these hemes; the C133Y mutation induced myxothiazol resistance, whereas the S206L did not modify the antimycin binding site, although perturbing the center N. These two amino acid replacements, along with those described elsewhere (Tron, T., and Lemesle-Meunier, D. (1990) Curr. Genet. 18, 413-419), constitute a novel class of mutants exhibiting appreciable electron transfer activity, despite their impaired ability to grow on respiratory substrates, raising the possibility that these mutants carry alleles which result in "decoupling" of proton translocation from electron transfer. Mutants W142R and M221K had an inactive but well assembled bc1 complex, whereas the G34OE and L282F mutations impaired the assembly of the bc1 complex.
    Preview · Article · Aug 1993 · Journal of Biological Chemistry

Publication Stats

119 Citations
29.76 Total Impact Points

Institutions

  • 1998
    • Massachusetts Institute of Technology
      • Department of Biology
      Cambridge, Massachusetts, United States
  • 1996
    • Geisel School of Medicine at Dartmouth
      • Department of Biochemistry
      Hanover, New Hampshire, United States
  • 1995
    • French National Centre for Scientific Research
      Lutetia Parisorum, Île-de-France, France