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JBIC Journal of Biological Inorganic Chemistry

, Volume 16, Issue 8, pp 1155–1168 | Cite as

Tyrosine phosphorylation turns alkaline transition into a biologically relevant process and makes human cytochrome c behave as an anti-apoptotic switch

  • José M. García-Heredia
  • Antonio Díaz-Quintana
  • Maria Salzano
  • Mar Orzáez
  • Enrique Pérez-Payá
  • Miguel Teixeira
  • Miguel A. De la Rosa
  • Irene Díaz-Moreno
Original Paper

Abstract

Cytochrome c (Cc) is a key protein in cell life (respiration) and cell death (apoptosis). On the one hand, it serves as a mitochondrial redox carrier, transferring electrons between the membrane-embedded complexes III and IV. On the other hand, it acts as a cytoplasmic apoptosis-triggering agent, forming the apoptosome with apoptosis protease-activating factor-1 (Apaf-1) and activating the caspase cascade. The two functions of cytochrome c are finely tuned by the phosphorylation of tyrosines and, in particular, those located at positions 48 and 97. However, the specific cytochrome c-phosphorylating kinase is still unknown. To study the structural and functional changes induced by tyrosine phosphorylation in cytochrome c, we studied the two phosphomimetic mutants Y48E and Y97E, in which each tyrosine residue is replaced by glutamate. Such substitutions alter both the physicochemical features and the function of each mutant compared with the native protein. Y97E is significantly less stable than the WT species, whereas Y48E not only exhibits lower values for the alkaline transition pK a and the midpoint redox potential, but it also impairs Apaf-1-mediated caspase activation. Altogether, these findings suggest that the specific phosphorylation of Tyr48 makes cytochrome c act as an anti-apoptotic switch.

Keywords

Apoptosis Human cytochrome c Peroxidase activity Post-translational modification Tyrosine phosphorylation 

Abbreviations

Ac-LEHD-AFC

N-acetyl-Leu-Glu-His-Asp-(7-amino-4-trifluoromethyl coumarin)

Apaf-1

Apoptosis protease-activating factor-1

BSA

Bovine serum albumin

CL

Cardiolipin

Cc

Cytochrome c

CcO

Cytochrome c oxidase

CD

Circular dichroism

DCF

2′,7′-Dichlorofluorescein

EPR

Electron paramagnetic resonance

H2DCF

Reduced 2′,7′-dichlorofluorescein

MD

Molecular dynamics

NMR

Nuclear magnetic resonance

PC9

Pro-caspase 9

RMSD

Root mean square deviation

RNOS

Reactive nitrogen/oxygen species

ROS

Reactive oxygen species

Tm

Midpoint melting temperature

Notes

Acknowledgments

The authors wish to thank the Spanish Ministry of Science and Innovation (BFU2009-07190) and the Andalusian Government (BIO198) for financial support. The authors declare that they have no competing financial interests.

Supplementary material

775_2011_804_MOESM1_ESM.pdf (669 kb)
Supplementary Material 1 (PDF 669 kb)

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Copyright information

© SBIC 2011

Authors and Affiliations

  • José M. García-Heredia
    • 1
  • Antonio Díaz-Quintana
    • 1
  • Maria Salzano
    • 1
  • Mar Orzáez
    • 2
  • Enrique Pérez-Payá
    • 2
    • 3
  • Miguel Teixeira
    • 4
  • Miguel A. De la Rosa
    • 1
  • Irene Díaz-Moreno
    • 1
  1. 1.Instituto de Bioquímica Vegetal y Fotosíntesis, cicCartujaUniversidad de Sevilla-CSICSevillaSpain
  2. 2.Centro de Investigación Príncipe FelipeValenciaSpain
  3. 3.Instituto de Biomedicina de Valencia, IBV-CSICValenciaSpain
  4. 4.Instituto de Tecnologia Química e BiológicaUniversidade Nova de LisboaOeirasPortugal

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