Skip to main content
SpringerLink
Log in

Cytochrome c is rapidly reduced in the cytosol after mitochondrial outer membrane permeabilization

  • Original Paper
  • Published:
Apoptosis Aims and scope Submit manuscript

Abstract

Visible spectroscopy was used to measure real-time changes in the oxidation state of cytochrome c (cyt c) and the a-cytochromes (cyt aa3) of cytochrome oxidase during mitochondrial outer membrane permeabilization (MOMP) initiated by anisomycin in HL-60 cells. The oxidation state of mitochondrial cyt c was found to be ≈62% oxidized before MOMP and became ≈70% oxidized after MOMP. In contrast, the cytosolic pool of cyt c was found to be almost fully reduced. This oxidation change allows cyt c release to be continuously and quantitatively monitored in real time. Anoxia and antimycin were used to fully reduce and fully oxidize, respectively, the mitochondrial pool of cyt c and it was found that the release of cyt c was independent of it oxidation state consistent with a simple model of cyt c passively diffusing down a concentration gradient through a pore or tear in the outer membrane. After MOMP was complete, the flux of cyt c diffusing back into the mitochondria was measured from the residual mitochondrial oxygen consumption after complete inhibition of the bc1 with antimycin and myxothiazol. The outer membrane was found to be highly permeable after MOMP implying that the reduction of cyt c in the cytosol must be very rapid. The permeability of the outer membrane measured in this study would result in the release of cyt c with a time constant of less than 1 s.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7
Fig. 8

Similar content being viewed by others

References

  1. Green DR, Kroemer G (2004) The pathophysiology of mitochondrial cell death. Science 305:626–629

    Article  PubMed  CAS  Google Scholar 

  2. Nieminen AI, Partanen JI, Klefstrom J (2007) c-Myc blazing a trail of death: coupling of the mitochondrial and death receptor apoptosis pathways by c-Myc. Cell Cycle 6:2464–2472

    PubMed  CAS  Google Scholar 

  3. Riedl SJ, Salvesen GS (2007) The apoptosome: signalling platform of cell death. Nat Rev Mol Cell Biol 8:405–413

    Article  PubMed  CAS  Google Scholar 

  4. 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  PubMed  CAS  Google Scholar 

  5. Rich PR, West IC, Mitchell P (1988) The location of CuA in mammalian cytochrome c oxidase. FEBS Lett 233:25–30

    Article  PubMed  CAS  Google Scholar 

  6. Chance B, Williams GR (1956) The respiratory chain and oxidative phosphorylation. Adv Enzymol Relat Areas Mol Biol 17:65–134

    Article  CAS  Google Scholar 

  7. Wilson DF, Rumsey WL, Green TJ, Vanderkooi JM (1988) The oxygen dependence of mitochondrial oxidative phosphorylation measured by a new optical method for measuring oxygen concentration. J Biol Chem 263:2712–2718

    PubMed  CAS  Google Scholar 

  8. Hollis VS, Palacios-Callender M, Springett RJ, Delpy DT, Moncada S (2003) Monitoring cytochrome redox changes in the mitochondria of intact cells using multi-wavelength visible light spectroscopy. Biochim Biophys Acta 1607:191–202

    Article  PubMed  CAS  Google Scholar 

  9. Hampton MB, Zhivotovsky B, Slater AF, Burgess DH, Orrenius S (1998) Importance of the redox state of cytochrome c during caspase activation in cytosolic extracts. Biochem J 329(1):95–99

    PubMed  CAS  Google Scholar 

  10. Pan Z, Voehringer DW, Meyn RE (1999) Analysis of redox regulation of cytochrome c-induced apoptosis in a cell-free system. Cell Death Differ 6:683–688

    Article  PubMed  CAS  Google Scholar 

  11. Suto D, Sato K, Ohba Y, Yoshimura T, Fujii J (2005) Suppression of the pro-apoptotic function of cytochrome c by singlet oxygen via a haem redox state-independent mechanism. Biochem J 392:399–406

    Article  PubMed  CAS  Google Scholar 

  12. Borutaite V, Brown GC (2007) Mitochondrial regulation of caspase activation by cytochrome oxidase and tetramethylphenylenediamine via cytosolic cytochrome c redox state. J Biol Chem 282:31124–31130

    Article  PubMed  CAS  Google Scholar 

  13. Matcher SJ, Cope M, Delpy DT (1994) Use of the water absorption spectrum to quantify tissue chromophore concentration changes in near-infrared spectroscopy. Phys Med Biol 39:177–196

    Article  PubMed  CAS  Google Scholar 

  14. Single B, Leist M, Nicotera P (1998) Simultaneous release of adenylate kinase and cytochrome c in cell death. Cell Death Differ 5:1001–1003

    Article  PubMed  CAS  Google Scholar 

  15. Ganju N, Eastman A (2002) Bcl-X(L) and calyculin A prevent translocation of Bax to mitochondria during apoptosis. Biochem Biophys Res Commun 291:1258–1264

    Article  PubMed  CAS  Google Scholar 

  16. Ow YL, Green DR, Hao Z, Mak TW (2008) Cytochrome c: functions beyond respiration. Nat Rev Mol Cell Biol 9:532–542

    Article  PubMed  CAS  Google Scholar 

  17. Goldstein JC, Waterhouse NJ, Juin P, Evan GI, Green DR (2000) The coordinate release of cytochrome c during apoptosis is rapid, complete and kinetically invariant. Nat Cell Biol 2:156–162

    Article  PubMed  CAS  Google Scholar 

  18. Bernardi P, Azzone GF (1981) Cytochrome c as an electron shuttle between the outer and inner mitochondrial membranes. J Biol Chem 256:7187–7192

    PubMed  CAS  Google Scholar 

  19. Capano M, Crompton M (2002) Biphasic translocation of Bax to mitochondria. Biochem J 367:169–178

    Article  PubMed  CAS  Google Scholar 

  20. Hinkle PC, Kumar MA, Resetar A, Harris DL (1991) Mechanistic stoichiometry of mitochondrial oxidative phosphorylation. Biochemistry 30:3576–3582

    Article  PubMed  CAS  Google Scholar 

  21. Atlante A, de Bari L, Bobba A, Marra E, Calissano P, Passarella S (2003) Cytochrome c, released from cerebellar granule cells undergoing apoptosis or excytotoxic death, can generate protonmotive force and drive ATP synthesis in isolated mitochondria. J Neurochem 86:591–604

    Article  PubMed  CAS  Google Scholar 

  22. Stennicke HR, Deveraux QL, Humke EW, Reed JC, Dixit VM, Salvesen GS (1999) Caspase-9 can be activated without proteolytic processing. J Biol Chem 274:8359–8362

    Article  PubMed  CAS  Google Scholar 

  23. Hancock JT, Desikan R, Neill SJ (2001) Does the redox status of cytochrome C act as a fail-safe mechanism in the regulation of programmed cell death? Free Radic Biol Med 31:697–703

    Article  PubMed  CAS  Google Scholar 

  24. Klatt P, Heinzel B, John M, Kastner M, Bohme E, Mayer B (1992) Ca2+/calmodulin-dependent cytochrome c reductase activity of brain nitric oxide synthase. J Biol Chem 267:11374–11378

    PubMed  CAS  Google Scholar 

  25. Fago A, Mathews AJ, Moens L, Dewilde S, Brittain T (2006) The reaction of neuroglobin with potential redox protein partners cytochrome b5 and cytochrome c. FEBS Lett 580:4884–4888

    Article  PubMed  CAS  Google Scholar 

  26. Murataliev MB, Feyereisen R, Walker FA (2004) Electron transfer by diflavin reductases. Biochim Biophys Acta 1698:1–26

    PubMed  CAS  Google Scholar 

  27. Finn RD, Basran J, Roitel O, Wolf CR, Munro AW, Paine MJ, Scrutton NS (2003) Determination of the redox potentials and electron transfer properties of the FAD- and FMN-binding domains of the human oxidoreductase NR1. Eur J Biochem 270:1164–1175

    Article  PubMed  CAS  Google Scholar 

  28. Al-Ayash AI, Wilson MT (1979) The mechanism of reduction of single-site redox proteins by ascorbic acid. Biochem J 177:641–648

    PubMed  CAS  Google Scholar 

  29. Everse J, Kujundzic N (1979) Kinetics and mechanism of the reduction of horse heart ferricytochrome c by glutathione. Biochemistry 18:2668–2673

    Article  PubMed  CAS  Google Scholar 

  30. Jiang S, Cai J, Wallace DC, Jones DP (1999) Cytochrome c-mediated apoptosis in cells lacking mitochondrial DNA. Signaling pathway involving release and caspase 3 activation is conserved. J Biol Chem 274:29905–29911

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgments

The project described was supported by Award Numbers R01NS054298 from the National Institute of Neurological Disorders and Stroke (NINDS) and R21RR025803 from the National Center for Research Resources (NCRR). The content is solely the responsibility of the authors and does not necessarily represent the official views of NINDS, NCRR or the National Institutes of Health.

Author information

Authors and Affiliations

  1. Department of Radiology, Dartmouth Medical School, HB7886 Vail, Hanover, NH, 03755, USA

    Maureen O. Ripple, Michelle Abajian & Roger Springett

Authors
  1. Maureen O. Ripple
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Michelle Abajian
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Roger Springett
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Roger Springett.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Ripple, M.O., Abajian, M. & Springett, R. Cytochrome c is rapidly reduced in the cytosol after mitochondrial outer membrane permeabilization. Apoptosis 15, 563–573 (2010). https://doi.org/10.1007/s10495-010-0455-2

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10495-010-0455-2

Keywords

Search

Navigation