CGM - Department Regulation and Compartmentalization of Cellular Functions - G. Dujardin's team
Function and dysfonction of respiratory complexes:
the cytochromes bc1 and oxidase
Principal Investigator: B. MEUNIER, avec C. Vallières
Last update: 28-Nov-2011
Theme
Our research focuses on the study of mitochondrial respiratory chain enzymes both from a basic point of view and as a tool in the agrochemical and biomedical fields. We study these enzymes using genetics, biophysics and biochemistry, in a model system: the yeast Saccharomyces cerevisiae. In particular, we analyze mitochondrial mutations, in cytochromes bc 1 and oxidase, which are either associated with human pathologies or with resistance to antimicrobial agents in plant or human pathogens.
The mitochondrial respiratory chain is composed of lipoprotein complexes embedded in the mitochondrial inner membrane. This chain of enzymes transfers electrons from NADH and FADH 2, that originate from various metabolic pathways, to oxygen. This transfer is coupled to the translocation of protons across the mitochondrial membrane. The proton gradient is then used for ATP synthesis by the ATP-synthase. This mechanism produces around 90% of the ATP in animal cells (Rich, P.R. The cost of living. Nature (2003) 421, 583).
Fig. 1 : The mitochondrial respiratory chain. Schematic representation of the respiratory chain in yeast.
NDH : NADH dehydrogenases; bc1 : cytochrome bc1 or complex III; SDH: succinate dehydrogenase or complex II; Cyt c: cytochrome c; CcO: cytochrome c oxidase or complex IV; ATP synthase or complex V.
The production of energy to maintain cellular metabolism is very demanding on the respiratory chain. Any defect in respiratory enzymes caused by genetic mutations or inhibitors will have disastrous consequences on cell survival.
We study two key enzymes of the respiratory chain: the cytochromes bc1 and oxidase. In particular, we analyze the impact of mitochondrial mutations that alter the catalytic core of these enzymes. The yeast Saccharomyces cerevisiae has been chosen as a model system for several reasons. First, yeast cells can survive in the absence of respiration by using the fermentation process as an energy source. Therefore the absence of one or more respiratory enzymes is not lethal. Second, it is easy to transform yeast mitochondria in order to introduce any desired mutation. Last, yeast mitochondrial enzymes and especially their catalytic cores are very similar to their human counterparts.
Resistance to antimicrobial agents that inhibit cytochrome bc1
Fig. 2 The catalytic core and the inhibitor binding site in cytochrome bc1.
Cytochrome c1, the Rieske protein and the mitochondrially – encoded cytochrome b form the catalytic core of the enzyme. Inhibitors, such as stigmatellin, bind in the quinol oxidation pocket (Qo). Modification of the Qo site could alter the binding of the inhibitor and therefore the sensitivity of the pathogen, or its yeast model, to treatment.
Respiration is essential to the survival and proliferation of certain microorganisms and has therefore been a target of choice for antimicrobial agents. During the last 10-15 years, cytochrome bc1 inhibitors have been developed as anti-microbial agents. They are used to control pathogenic fungi in plants, and in medicine, in treatments against infectious agents like Plasmodium falciparum , the malaria agent or Pneumocystis jiroveci, an opportunistic pathogenic fungus causing life-threatening pneumonia in immunodeficient patients. These inhibitors are active against a large spectrum of pathogens. Unfortunately, an increasing number of acquired resistances toward the treatments has been observed, often associated with mutations in cytochrome b, the main subunit of the enzyme. We have transferred these mutations in the yeast system to study their impact on the sensitivity to the drugs, the respiratory capacity and the fitness of yeast.
These analyses should allow us to better understand the molecular basis of resistance and therefore contribute to the study and prediction of the evolution of resistance in the pathogen populations.
Fig. 3 Mitochondrial genes and diseases.
We are currently studying ‘disease’ mutations in the genes for cytochrome b and for the subunits 1, 2 and 3 of cytochrome oxidase.
Human mitochondrial mutations
In humans, mitochondrial diseases are the most frequent metabolic diseases. More than 100 mutations have been reported in mitochondrial genes (http://www.mitomap.org/). These genes encode subunits of the respiratory enzymes cytochrome oxidase, cytochrome bc1 and ATP-synthase, as well as proteins of the translation apparatus.
We have introduced some of these mutations in cytochrome b and in the mitochondrial subunits of cytochrome oxidase into yeast, in order to study their impact at the structural and mechanistic levels. We are studying their effects on the assembly of the respiratory complexes, the spectral properties of redox centers and the enzymatic activities. In parallel, we also study the sequence variations observed in asymptomatic individuals, which could affect the respiratory mechanism.
Improvement of the model system
We have undertaken the remodeling of the catalytic site of yeast cytochrome b (the so called Qo site) by replacing residues specific to yeast by residues found in humans. This model, closer to the human situation, should prove more useful to study the impact of mutations and the inhibitor specificities.
Current collaborations
- G. Brasseur, Interactions et Modulateurs de Réponses, IBSM-CNRS, Marseille: web site
- N. Fisher, Liverpool School of Tropical Medicine, UK: web site
- S. Rahman, Mitochondrial Research Group, Clinical and Molecular Genetics, Institute of Child Health, London, UK: web site
- F. Rappaport, Institut de Biologie Physico-Chimique, Paris, France: web site
- P. Rich, Institute of Structural and Molecular Biology, UCL, London, UK: web site
- B. Trumpower, Dartmouth Medical School, Hanover, USA: web site
- D. Winge, University of Utah, USA: web site
Publications since 2003
Maréchal, A., Meunier, B., Lee, D., Orengo, C., Rich, PR. (2011) Yeast cytochrome c oxidase: A model system to study mitochondrial forms of the haem-copper oxidase superfamily. Biochim Biophys Acta, Epub ahead of print.
Trouillard, M., Meunier, B., Rappaport, F. (2011) Questioning the functional relevance of mitochondrial supercomplexes by time-resolved analysis of the respiratory chain. Proc Natl Acad Sci U S A., 108 (45) E1027-34.
Glatigny, A., Mathieu, L., Herbert, CJ., Dujardin, G., Meunier, B., Mucchielli-Giorgi, MH. (2011) An in silico approach combined with in vivo experiments enables the identification of a new protein whose overexpression can compensate for specific respiratory defects in Saccharomyces cerevisiae. BMC Syst Biol, 5 (1) 173.
Vallières, C., Trouillard, M., Dujardin, G., Meunier, B. (2011) Studying the deleterious effect of the QoI resistance mutation G143A in the intron-containing cytochrome b gene and the by-pass mechanisms. Appl Environ Microbiol, 77 (6) 2088-93.
Khalimonchuk, O., Bestwick, M., Meunier, B., Watts, T.-C. and Winge, D.-R. (2010) Formation of the redox cofactor centers during Cox1 maturation in yeast cytochrome oxidase. Mol Cell Biol, 30 (4) 1004-17.
Duncan, A., Bitner-Glindzicz, M., Meunier, B., Costello, H., Hargreaves, I., Lopez, L., Hirano, M., Sadowski, M. I., Hardy, J., Singleton, A., Clayton, P. and Rahman, S. (2009) A nonsense mutation in COQ9 causes autosomal recessive neonatal onset primary coenzyme Q10 deficiency – a potentially treatable form of mitochondrial disease. Am J Hum Genet, 84 (5) 558-66.
Bourges, I., Mucchielli, M.-H., Herbert, C.-J., Guiard, B., Dujardin, G. and Meunier, B. (2009) Multiple Defects in the Respiratory Chain Lead to the Repression of Genes Encoding Components of the Respiratory Chain and TCA Cycle Enzymes. J Mol Biol, 23 (1) 23-42.
Biagini, G., Fisher, N., Berry, N., Stocks, P., Meunier, B., Williams, D., Bonar-Law, R., Bray, P., Owen, A., O'Neill, P. and Ward, S. (2008) Acridinediones: Selective and Potent Inhibitors of the Malaria Parasite Mitochondrial bc1 Complex. Mol Pharmacol 73 (5) 1347-1355.
Fisher, N. and Meunier, B. (2008) Molecular basis of resistance to cytochrome bc1 inhibitors. FEMS Yeast Res 8 (2) 183-192.
Seddiki, N., Meunier, B., Lemesle-Meunier, D. and Brasseur, G. (2008) Is cytochrome b glutamic acid 272 a quinol binding residue in the bc1 complex of Saccharomyces cerevisiae? Biochemistry, 47 (8) 2357-68.
Wenz, T., Covian, R., Hellwig, P., MacMillan, F., Meunier, B.; Trumpower, B. L. and Hunte, C. J. Biol. Chem. (2007) 282, 3977-88. Mutational analysis of cytochrome b at the ubiquinol oxidation site of yeast complex III.
Wenz, T., Hellwig, P., MacMillan, F., Meunier, B. and Hunte, C. (2006) Biochemistry. 45, 9042-9052. Probing the role of E272 in quinol oxidation of mitochondrial complex III.
Pye, D., Kyriakouli, D.S., Taylor, G., Taha, R., Elstner, M., Meunier, B., Chrzanowska-Lightowlers, Z.M.A., Taylor, R.W., Turnbull, D.M. and Lightowlers, R.N. (2006) Nucl. Acids Res. 34 (13), e95. Production of transmitochondrial cybrids containing naturally occurring pathogenic mtDNA variants.
Horan, S., Bourges, I. and Meunier, B. (2006) YEAST. 23, 519-535. Transcriptional response to nitrosative stress in Saccharomyces cerevisiae.
Bourges, I., Horan, S., and Meunier, B. (2005) J. Biol. Chem. 280, 29743 - 29749. Effect of inhibition of the bc1 complex on nuclear gene expression profile in yeast
Kessl, J.J., Ha, K. H., Merritt, A.K., Lange, B. B., Hill, P., Meunier, B., Meshnick, S. and Trumpower, B. L. (2005) J. Biol. Chem. 280, 17142-17148. Cytochrome b mutations that modify the ubiquinol-binding pocket of the cytochrome bc1 complex and confer anti-malarial drug resistance in Saccharomyces cerevisiae.
Fisher, N. and Meunier, B. (2005) Pest Manag. Sci. 61, 973-978. Re-examination of inhibitor resistance conferred by Qo-site mutations in cytochrome b using yeast as a model system.
Blakely, E.L., Mitchell, A.L., Fisher, N., Meunier, B., Nijtmans, L.G., Schaefer A.M., Jackson, M.J., Turnbull, D.M. and Taylor, R.W. (2005) FEBS J. 272, 3583-3592. A pathogenic mitochondrial MTCYB mutation (Arg318Pro) causing severe respiratory chain enzyme deficiency: studies in human and yeast
Horan, S., Bourges, I., Taanman, J.W. and Meunier, B. (2005) Biochem. J. 390, 703-708. Analysis of COX2 mutants reveals cytochrome oxidase subassemblies in yeast
Brasseur, G., Lemesle-Meunier, D., Reinaud, F. and Meunier, B. (2004) J. Biol. Chem . 279 (23) 34203-24211. Qo site deficiency can be compensated by extragenic mutations in the hinge region of the Rieske protein in the bc1 complex of Saccharomyces cerevisiae.
Fisher, N., Castleden, C. K., Bourges, I., Brasseur, G., Dujardin, G. and Meunier, B. (2004) J. Biol. Chem. 279, 12951-12958. Disease-related mutations in cytochrome b studied in yeast.
Kessl, J.J., Hill, P., Lange, B. B., Meshnick, S., Meunier, B. and Trumpower, B. L. (2004) J. Biol. Chem. 279, 2817-2824. Molecular basis for atovaquone resistance in Pneumocystis carinii modelled in cytochrome bc1 complex of Saccharomyces cerevisiae.
Fisher, N., Brown, A. C., Sexton, G., Cook, A., Windass, J. and Meunier, B. (2004) Eur. J. Biochem. 271, 2264-2271. Modelling the Qo site of crop pathogens in Saccharomyces cerevisiae cytochrome b.
Fisher, N., Bourges, I., Hill, P., Brasseur, G. and Meunier, B. (2004) Eur. J. Biochem. 271, 1292-1298. Disruption of the interaction between the Rieske iron-sulphur protein and cytochrome b in yeast bc1 complex owing to a human disease-associated mutation within cytochrome b.
Bratton, M., Denise Mills, D., Castleden, C. K., Hosler, J. and Meunier, B. (2003) Eur. J. Biochem. 270, 1222-1230. Disease-related mutations in cytochrome c oxidase studied in yeast and bacterial models.
Kessl, J.J., Lange, B. B., Merbitz-Zahradnik, T., Zwicker, K., Hill, P., Meunier, B., Meshnick, S., Trumpower, B. L. (2003) J. Biol. Chem. 278, 31321-31318. Molecular basis for atovaquone binding to the cytochrome bc1 complex.
Hill, P., Kessl, J., Fisher, N., Meshnick, S., Trumpower, B. L. and Meunier, B. (2003). Antimicrob. Agents Chemother. 47, 2725-2731. Recapitulation in Saccharomyces cerevisiae of cytochrome b mutations conferring resistance to atovaquone in Pneumocystis jiroveci.
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