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

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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.

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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

T m :

Midpoint melting temperature

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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.

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Authors and Affiliations

  1. Instituto de Bioquímica Vegetal y Fotosíntesis, cicCartuja, Universidad de Sevilla-CSIC, Americo Vespucio 49, 41092, Sevilla, Spain

    José M. García-Heredia, Antonio Díaz-Quintana, Maria Salzano, Miguel A. De la Rosa & Irene Díaz-Moreno

  2. Centro de Investigación Príncipe Felipe, Autopista del Saler 16, 46012, Valencia, Spain

    Mar Orzáez & Enrique Pérez-Payá

  3. Instituto de Biomedicina de Valencia, IBV-CSIC, Jaume Roig, Valencia, Spain

    Enrique Pérez-Payá

  4. Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Avenida da República, 2780-157, Oeiras, Portugal

    Miguel Teixeira

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  1. José M. García-Heredia
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  2. Antonio Díaz-Quintana
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Correspondence to Irene Díaz-Moreno.

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García-Heredia, J.M., Díaz-Quintana, A., Salzano, M. et al. Tyrosine phosphorylation turns alkaline transition into a biologically relevant process and makes human cytochrome c behave as an anti-apoptotic switch. J Biol Inorg Chem 16, 1155–1168 (2011). https://doi.org/10.1007/s00775-011-0804-9

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