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Study of Respiratory Cytochromes in Liposomes

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Liposomes

Part of the book series: Methods in Molecular Biology™ ((MIMB,volume 606))

Abstract

Important findings regarding the structure and function of respiratory cytochromes have been made from the study of these hemeproteins associated to liposomes. These studies contributed to the comprehension of the biological role of these proteins in the electron transfer process, the regulatory mechanisms, the energy transduction mechanisms, the protein sites that interact with mitochondrial membranes and the role played by the non-redox subunits present in the protein complexes of the respiratory chain of eukaryotes. In this chapter, the protocols developed to study cytochrome bc 1 activity in liposomes and the binding of cytochrome c to lipid bilayers is presented . The former protocol was developed to study the mechanism of energy transduction related to the topology of the components of bc 1 complex in the mitochondrial membrane. These studies were done with purified cytochrome bc 1 complexes reconstituted into potassium-loaded vesicles. The latter protocol was developed to study the influence of pH, ΔpH, and ΔΨ on the interaction of cytochrome c with liposomes that mimic the inner mitochondrial membrane.

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References

  1. Trumpower BL (1990) The protonmotive Q cycle. J Biol Chem 265:11409-11412

    CAS  PubMed  Google Scholar 

  2. Boyer PD (1993) The binding change mechanism for ATP synthase. Some probabilities and possibilities. Biochim Biophys Acta 1440:215-250

    Google Scholar 

  3. Erecinska M, Wilson DF (1982) Regulation of cellular energy metabolism. J Membr Biol 70:1-14

    Article  CAS  PubMed  Google Scholar 

  4. Mitchell P (1976) Possible molecular mechanisms of the protonmotive function of cytochrome systems. J Theor Biol 62:327-367

    Article  CAS  PubMed  Google Scholar 

  5. Robertson DE, Dutton PL (1988) The nature and magnitude of the charge-separation reactions of ubiquinol cytochrome c2 oxidoreductase. Biochim Biophys Acta 935:273-291

    Article  CAS  PubMed  Google Scholar 

  6. Drachev LA, Kaurov BS, Mamedov MD, Mulkidjanian AY, Semonev AY, Shinkarev VP, Skulachev VP, Verkhovsky MI (1989) Biochim Biophys Acta 973:189-197

    Google Scholar 

  7. Konstantinov A, Kunz WS, Kamensky YA (1981) In: Skulachev VP, Hinkle PC (eds) Chemiosmotic proton circuits in biological membranes, Addison-Wesley pp 123-146

    Google Scholar 

  8. Miki T, Mikin M, Orii Y (1994) Membrane potential-linked reversed electron transfer in the Beef Heart Cytochrome bcl complex reconstituted into potassium-loaded phospholipid vesicles. J Biol Chem 269:1827-1833

    CAS  PubMed  Google Scholar 

  9. West IC, Mitchell P, Rich PR (1988) Electron conduction between b cytochromes of the mitochondrial respiratory chain in the presence of antimycin plus myxothiazol. Biochim Biophys Acta 933:35-41

    Article  CAS  PubMed  Google Scholar 

  10. Konstantinov AA, Popova E (1988) In: Papa S (ed) Cytochrome systems: molecular biology and bioenergetics, Plenum Publishing Corp., New York, pp 751-765

    Google Scholar 

  11. Kamensky YuA, Artzabanov VYu, Shevchenko DV, Konstantinov AA (1979) Effect of antimycin A on redox-dependent protonation of the b cytochromes of the mitochondrial respiratory chain. Dokl Acad Nauk SSSR 249:994-997

    Google Scholar 

  12. Von Jagow G, Link TA, Ohnishi T (1986) Organization and function of cytochrome b and ubiquinone in the cristae membrane of beef heart mitochondria. J Bioenerg Biomembr 18:157-179

    Article  Google Scholar 

  13. Kluck RM, BossyWetzel E, Green DR, Newmeyer DD (1997) Science 275:1132-1136

    Google Scholar 

  14. Yang J, Liu XS, Bhalla K, Kim CN, Ibrado AM, Cai J, Peng T, Jones DP, Wang X (1997) Science 275:1129-1132

    Google Scholar 

  15. Crompton M (1999) The mitochondrial permeability transition pore and its role in cell death. Biochem J 341:233-249

    Article  CAS  PubMed  Google Scholar 

  16. Lenaz G (1998) Quinone specificity of complex. Biochim Biophys Acta 1364:207-221

    Article  CAS  PubMed  Google Scholar 

  17. De Giorgi F, Lartigue L, Bauer MK, Schubert A, Grimm S, Hanson GT, Remington SJ, Youle RJ, Ichas F (2002) The permeability transition pore signals apoptosis by directing Bax translocation and multimerization. FASEB J 16:607-609

    PubMed  Google Scholar 

  18. Matroule JY, Carthy CM, Granville DJ, Jolois O, Hunt DW, Piette J (2001) Mechanism of colon cancer cell apoptosis mediated by pyropheophorbide-a methylester photosensitization. Oncogene 20:4070-4084

    Article  CAS  PubMed  Google Scholar 

  19. Sugawara T, Lewen A, Gasche Y, Yu F, Chan PH (2002) Overexpression of SOD1 protects vulnerable motor neurons after spinal cord injury by attenuating mitochondrial cytochrome c release. FASEB J 16:1997-1999

    CAS  PubMed  Google Scholar 

  20. Kowaltowski AJ, Castilho RF, Vercesi AE (2001) Mitochondrial permeability transition and oxidative stress. FEBS Lett 495:12-15

    Article  CAS  PubMed  Google Scholar 

  21. Kowaltowski AJ, Vercesi AE, Fiskum G (2000) Bcl-2 prevents mitochondrial permeability transition and cytochrome c release via maintenance of reduced pyridine nucleotides. Cell Death Differ 7:903-910

    Article  CAS  PubMed  Google Scholar 

  22. Kowaltowski AJ, Vercesi AE (1999) Mitochondrial damage induced by conditions of oxidative stress. Free Radic Biol Med 26:463-471

    Article  CAS  PubMed  Google Scholar 

  23. Barros MH, Netto LE, Kowaltowski AJ (2003) H2O2 generation in Saccharomyces cerevisiae respiratory pet mutants: effect of cytochrome c. Free Radic Biol Med 35:179-188

    Article  CAS  PubMed  Google Scholar 

  24. Kowaltowski AJ, Turin J, Indig GL, Vercesi AE (1999) Mitochondrial effects of triarylmethane dyes. J Bioenerg Biomembr 31:581-590

    Article  CAS  PubMed  Google Scholar 

  25. Wuthrich K, Aviram I, Schejter A (1971) Structural studies of modified cytochromes c by nuclear magnetic resonance spectroscopy. Biochim Biophys Acta 253:98-103

    Article  CAS  PubMed  Google Scholar 

  26. Nicholls P (1974) Cytochrome c binding to enzymes and membranes. Biochim Biophys Acta 346:261-310

    CAS  PubMed  Google Scholar 

  27. Pinheiro TJ (1994) The interaction of horse heart cytochrome c with phospholipid bilayers. Structural and dynamic effects. Biochimie 76:489-500

    Article  CAS  PubMed  Google Scholar 

  28. Nantes IL, Zucchi MR, Nascimento OR, Faljoni-Alario A (2001) Effect of heme iron valence state on the conformation of cytochrome c and its association with membrane interfaces. A CD and EPR investigation. J Biol Chem 276:153-158

    Article  CAS  PubMed  Google Scholar 

  29. Rytömaa M, Kinnunen PK (1995) Reversibility of the binding of cytochrome c to liposomes. Implications for lipid-protein interactions. J Biol Chem 270:3197-3202

    Article  PubMed  Google Scholar 

  30. Tuominen EK, Wallace CJ, Kinnunen PK (2002) Phospholipid-cytochrome c interaction: evidence for the extended lipid anchorage. J Biol Chem 277:8822-8826

    Article  CAS  PubMed  Google Scholar 

  31. Zucchi MR, Nascimento OR, Faljoni-Alário A, Prieto T, Nantes IL (2003) Modulation of cytochrome c spin states by lipid acyl chains: a continuous-wave electron paramagnetic resonance (CW-EPR) study of haem iron. Biochem J 370:671-678

    Article  CAS  PubMed  Google Scholar 

  32. Rytömaa M, Mustonen P, Kinnunen PKJ (1992) Reversible, nonionic, and pH-dependent association of cytochrome c with cardiolipin-phosphatidylcholine liposomes. J Biol Chem 267:22243-22248

    PubMed  Google Scholar 

  33. Rytömaa M, Kinnunen PKJ (1994) Evidence for two distinct acidic phospholipid-binding sites in cytochrome c. J Biol Chem 269:1770-1774

    PubMed  Google Scholar 

  34. Pelletier H, Kraut J (1992) Crystal structure of a complex between electron transfer partners, cytochrome c peroxidase and cytochrome c. Science 258:1748-1755

    Article  CAS  PubMed  Google Scholar 

  35. Hatefi Y, Rieske JS (1967) Methods Enzymol 10:225-230

    Article  CAS  Google Scholar 

  36. Margoliash E, Frohwirt N (1959) Spectrum of horse-heart cytochrome c. Biochem J 71:570-572

    CAS  PubMed  Google Scholar 

  37. Sone N, Yoshida M, Hirata H, Kagawa Y (1977) J Biochem (Tokyo) 81:519-528

    Google Scholar 

  38. Kawai C, Prado FM, Nunes GLC, Di Mascio P, Carmona-Ribeiro AM, Nantes IL (2005) pH-dependent interaction of cytochrome c with mitochondrial mimetic membranes. The role of an array of positively charged amino acids. J Biol Chem 280:34709-34717

    Article  CAS  PubMed  Google Scholar 

  39. Margoliash E, Frohwirt N (1959) Spectrum of horse-heart cytochrome c. Biochem J 71:570-572

    CAS  PubMed  Google Scholar 

  40. Bartlett GR (1959) Phosphorus assay in the column chromatography. J Biol Chem 234:466-468

    CAS  PubMed  Google Scholar 

  41. King TE (1961) J Biol Chem 236:2342-2346

    CAS  PubMed  Google Scholar 

  42. Yu CA, Yu L, King TE (1974) J Biol Chem 249:4905-4910

    CAS  PubMed  Google Scholar 

  43. Yu CA, Yu L (1980) Resolution and reconstitution of succinate-cytochrome c reductase: preparations and properties of high purity succinate dehydrogenase and ubiquinol-cytochrome c reductase. Biochim Biophys Acta 591:409-420

    Article  CAS  PubMed  Google Scholar 

  44. Leung KH, Hinkle PC (1975) Reconstitution of ion transport and respiratory control in vesicles formed from reduced coenzyme Q-cytochrome c reductase and phospholipids. J Biol Chem 250:8467-8471

    CAS  PubMed  Google Scholar 

  45. Beattie DS, Villalobo A (1982) Energy transduction by the reconstituted b-c1 complex from yeast mitochondria. Inhibitory effects of dicyclohexylcarbodiimide. J Biol Chem 257:14745-14752

    CAS  PubMed  Google Scholar 

  46. Miki T, Orii Y, Mukohata Y (1987) A mechanism of respiratory control: studies with proteoliposomes containing cytochrome oxidase and bacteriorhodopsin. J Biochem 102:199-209

    CAS  PubMed  Google Scholar 

  47. Gutweniger H, Massari S, Beltrame M, Colonna R, Veronesse P, Ziche B (1977) Biochim Biophys Acta 459:216-224

    Google Scholar 

  48. Miki T, Orii Y (1986) Cytochrome c peroxidase activity of bovine heart cytochrome oxidase incorporated in liposomes and generation of membrane potential. J Biochem 100:735-745

    CAS  PubMed  Google Scholar 

  49. Redfeam ER (1967) Isolation and determination of ubiquinone. Methods Enzymol 10:381-384

    Article  Google Scholar 

  50. Tsai A-L, Olson JS, And Palmer G (1987) The kinetics of reoxidation of yeast complex III. An evaluation of the Q-cycle. J Biol Chem 262:8677-8684

    CAS  PubMed  Google Scholar 

  51. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein measurement with the Folin phenol reagent. J Biol Chem 193:265-275

    CAS  PubMed  Google Scholar 

  52. Kawai C, Prado FM, Nunes GLC, Di Mascio P, Carmona-Ribeiro AM, Nantes IL (2005) pH-dependent interaction of cytochrome c with mitochondrial mimetic membranes. The role of an array of positively charged amino acids. J Biol Chem 280:34709-34717

    Article  CAS  PubMed  Google Scholar 

  53. Hunter DG, Frisken BJ (1998) Effect of extrusion pressure and lipid properties on the size and polydispersity of lipid vesicles. Biophys J 74:2996-3002

    Article  CAS  PubMed  Google Scholar 

  54. Nichols JW, Deamer DW (1980) Net proton-hydroxyl permeability of large unilamellar liposomes measured by an acid-base titration technique. Proc Natl Acad Sciences (USA) 77:2038-2042

    Article  CAS  Google Scholar 

  55. Handbook of Chemistry and Physics (1983) part II

    Google Scholar 

  56. Schenkman S, Araujo PS, Dijkman R, Qina FH, Chaimovich H (1981) Effects of temperature and lipid composition on the serum albumin-induced aggregation and fusion of small unilamellar vesicles. Biochm Biophy Acta 649:633-641

    Article  CAS  Google Scholar 

  57. Carmona-Ribeiro AM, Yoshida LS, Chaimovich H (1985) Salt effects on the stability of dioctadecyldimethylammonium chloride and sodium dihexadecyl phosphate vesicles. J Phys Chem 89:2928-2933

    Article  CAS  Google Scholar 

  58. Grabowski EF, Morrison ID (1983) Particle size distributions from analyses of quase-elastic light-scattering data. In: Dahneke B (ed) Measurements of suspended particles by quase-elastic light-scattering, Wiley-Interscience, NY, p 7

    Google Scholar 

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Correspondence to Iseli L. Nantes .

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© 2010 Humana Press, a part of Springer Science+Business Media, LLC

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Nantes, I.L., Kawai, C., Pessoto, F.S., Mugnol, K.C.U. (2010). Study of Respiratory Cytochromes in Liposomes. In: Weissig, V. (eds) Liposomes. Methods in Molecular Biology™, vol 606. Humana Press. https://doi.org/10.1007/978-1-60761-447-0_12

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  • DOI: https://doi.org/10.1007/978-1-60761-447-0_12

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  • Publisher Name: Humana Press

  • Print ISBN: 978-1-60761-446-3

  • Online ISBN: 978-1-60761-447-0

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