Skip to main content
PMC home page
Biochemical Journal logoLink to Biochemical Journal
. 1998 Jan 1;329(Pt 1):95–99. doi: 10.1042/bj3290095

Importance of the redox state of cytochrome c during caspase activation in cytosolic extracts.

M B Hampton 1, B Zhivotovsky 1, A F Slater 1, D H Burgess 1, S Orrenius 1
PMCID: PMC1219018  PMID: 9405280

Abstract

The export of cytochrome c from mitochondria to the cytoplasm has been detected during apoptosis. Addition of cytochrome c to cytosolic extracts can activate the caspases, suggesting that this export could be an important intracellular signal for initiating the apoptotic programme. We have investigated the mechanism of caspase activation by cytochrome c. Mitochondrial cytochrome c normally shuttles electrons between complexes III and IV of the electron transport chain. Interaction with these complexes is dependent on electrostatic interactions via a polylysine binding pocket. Cytosolic caspase activation was only observed with intact holocytochrome c, and increasing the ionic composition of the extracts prevented activation, suggesting that stringent allosteric interactions between cytochrome c and other cytoplasmic factors are necessary. Cytochrome c was fully reduced within 5 min of addition to the cytosolic extracts. Potassium ferricyanide could maintain cytochrome c in an oxidized state, but care was taken to use ferricyanide at concentrations where its polyanion effect did not cause interference. The oxidized form of cytochrome c was able to activate the caspases. We conclude that reduced cytochrome c will function in the cytoplasm; however, its reduction is not a critical step, and electron transfer from cytochrome c to its cytoplasmic-binding partner(s) is not necessary in the pathway leading to apoptosis.

Full Text

The Full Text of this article is available as a PDF (347.0 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Cohen G. M. Caspases: the executioners of apoptosis. Biochem J. 1997 Aug 15;326(Pt 1):1–16. doi: 10.1042/bj3260001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Craig D. B., Wallace C. J. ATP binding to cytochrome c diminishes electron flow in the mitochondrial respiratory pathway. Protein Sci. 1993 Jun;2(6):966–976. doi: 10.1002/pro.5560020610. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Duan H., Chinnaiyan A. M., Hudson P. L., Wing J. P., He W. W., Dixit V. M. ICE-LAP3, a novel mammalian homologue of the Caenorhabditis elegans cell death protein Ced-3 is activated during Fas- and tumor necrosis factor-induced apoptosis. J Biol Chem. 1996 Jan 19;271(3):1621–1625. doi: 10.1074/jbc.271.3.1621. [DOI] [PubMed] [Google Scholar]
  4. Fisher W. R., Taniuchi H., Anfinsen C. B. On the role of heme in the formation of the structure of cytochrome c. J Biol Chem. 1973 May 10;248(9):3188–3195. [PubMed] [Google Scholar]
  5. Kluck R. M., Bossy-Wetzel E., Green D. R., Newmeyer D. D. The release of cytochrome c from mitochondria: a primary site for Bcl-2 regulation of apoptosis. Science. 1997 Feb 21;275(5303):1132–1136. doi: 10.1126/science.275.5303.1132. [DOI] [PubMed] [Google Scholar]
  6. Kroemer G., Zamzami N., Susin S. A. Mitochondrial control of apoptosis. Immunol Today. 1997 Jan;18(1):44–51. doi: 10.1016/s0167-5699(97)80014-x. [DOI] [PubMed] [Google Scholar]
  7. Liu X., Kim C. N., Yang J., Jemmerson R., Wang X. Induction of apoptotic program in cell-free extracts: requirement for dATP and cytochrome c. Cell. 1996 Jul 12;86(1):147–157. doi: 10.1016/s0092-8674(00)80085-9. [DOI] [PubMed] [Google Scholar]
  8. Margalit R., Schejter A. Cytochrome c: a thermodynamic study of relationships among oxidation state, ion-binding and structural parameters. 2. Ion-binding linked to oxidation state. Eur J Biochem. 1973 Feb 1;32(3):500–505. doi: 10.1111/j.1432-1033.1973.tb02634.x. [DOI] [PubMed] [Google Scholar]
  9. McIntosh D. B., Parrish J. C., Wallace C. J. Definition of a nucleotide binding site on cytochrome c by photoaffinity labeling. J Biol Chem. 1996 Aug 2;271(31):18379–18386. doi: 10.1074/jbc.271.31.18379. [DOI] [PubMed] [Google Scholar]
  10. Nicholson D. W., Ali A., Thornberry N. A., Vaillancourt J. P., Ding C. K., Gallant M., Gareau Y., Griffin P. R., Labelle M., Lazebnik Y. A. Identification and inhibition of the ICE/CED-3 protease necessary for mammalian apoptosis. Nature. 1995 Jul 6;376(6535):37–43. doi: 10.1038/376037a0. [DOI] [PubMed] [Google Scholar]
  11. Nisimoto Y., Otsuka-Murakami H., Iwata S., Isogai Y., Iizuka T. Characterization of superoxide dismutase-insensitive cytochrome c reductase activity in HL-60 cytosol as NADPH-cytochrome P450 reductase. Arch Biochem Biophys. 1993 May;302(2):315–321. doi: 10.1006/abbi.1993.1217. [DOI] [PubMed] [Google Scholar]
  12. Wallach D. Apoptosis. Placing death under control. Nature. 1997 Jul 10;388(6638):123, 125-6. doi: 10.1038/40516. [DOI] [PubMed] [Google Scholar]
  13. Yang J., Liu X., Bhalla K., Kim C. N., Ibrado A. M., Cai J., Peng T. I., Jones D. P., Wang X. Prevention of apoptosis by Bcl-2: release of cytochrome c from mitochondria blocked. Science. 1997 Feb 21;275(5303):1129–1132. doi: 10.1126/science.275.5303.1129. [DOI] [PubMed] [Google Scholar]
  14. Zhivotovsky B., Burgess D. H., Vanags D. M., Orrenius S. Involvement of cellular proteolytic machinery in apoptosis. Biochem Biophys Res Commun. 1997 Jan 23;230(3):481–488. doi: 10.1006/bbrc.1996.6016. [DOI] [PubMed] [Google Scholar]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES