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Structural basis of mitochondrial dysfunction in response to cytochrome c phosphorylation at tyrosine 48

  1. Irene Díaz-Morenoa,2
  1. aInstituto de Investigaciones Químicas, cicCartuja, Universidad de Sevilla–Spanish National Scientific Council (CSIC), 41092 Seville, Spain;
  2. bMagnetic Resonance Center (CERM), Department of Chemistry, University of Florence, 50019 Sesto Fiorentino, Florence, Italy;
  3. cDiamond Light Source, Didcot, Oxfordshire OX11 0DE, United Kingdom;
  4. dCentro Andaluz de Biología del Desarrollo, Universidad Pablo de Olavide–CSIC, 41013 Seville, Spain;
  5. eInstitute of Biocomputation and Physics of Complex Systems (BIFI), Joint Unit BIFI-Instituto Química-Física Rocasolano (CSIC), Universidad de Zaragoza, 50018 Zaragoza, Spain

  1. Edited by Kara L. Bren, University of Rochester, Rochester, NY, and accepted by Editorial Board Member Harry B. Gray March 8, 2017 (received for review November 1, 2016)

Significance

Cell response to physiological changes and oxidative stress involves the modulation of mitochondrial metabolism. Its dysfunction favors the development of hypoxia-dependent pathologies, including ischemia and cancer. A key modulator of mitochondrial activity is cytochrome c, whose cell function is regulated by tyrosine phosphorylation. However, how such modification affects cytochrome c structure and function is barely known. Here we report that a phosphomimetic mutant of cytochrome c exhibits enhanced dynamics, which could be responsible for the observed differences in cytochrome c functionality in oxidative stress and cell death. Thus, phosphorylation of cytochrome c becomes a target for further development of robust therapeutic approaches.

Abstract

Regulation of mitochondrial activity allows cells to adapt to changing conditions and to control oxidative stress, and its dysfunction can lead to hypoxia-dependent pathologies such as ischemia and cancer. Although cytochrome c phosphorylation—in particular, at tyrosine 48—is a key modulator of mitochondrial signaling, its action and molecular basis remain unknown. Here we mimic phosphorylation of cytochrome c by replacing tyrosine 48 with p-carboxy-methyl-l-phenylalanine (pCMF). The NMR structure of the resulting mutant reveals significant conformational shifts and enhanced dynamics around pCMF that could explain changes observed in its functionality: The phosphomimetic mutation impairs cytochrome c diffusion between respiratory complexes, enhances hemeprotein peroxidase and reactive oxygen species scavenging activities, and hinders caspase-dependent apoptosis. Our findings provide a framework to further investigate the modulation of mitochondrial activity by phosphorylated cytochrome c and to develop novel therapeutic approaches based on its prosurvival effects.

Footnotes

  • ↵1B.M.-B. and A.G.-C. contributed equally to this work.

  • ↵2To whom correspondence should be addressed. Email: idiazmoreno{at}us.es.

  • Author contributions: B.M.-B., A.G.-C., A.D.-Q., R.D.C., S.D.-M., C.S.-O., A.V.-C., M.A.R., P.T., and I.D.-M. designed research; B.M.-B., A.G.-C., A.D.-Q., R.D.C., S.M.G.-M., S.D.-M., K.G.-A., C.S.-O., A.V.-C., P.T., and I.D.-M. performed research; I.D.-M. contributed new reagents/analytic tools; B.M.-B., A.G.-C., A.D.-Q., R.D.C., A.V.-C., P.T., and I.D.-M. analyzed data; and B.M.-B., A.G.-C., A.D.-Q., M.A.R., P.T., and I.D.-M. wrote the paper.

  • The authors declare no conflict of interest.

  • This article is a PNAS Direct Submission. K.L.B. is a Guest Editor invited by the Editorial Board.

  • Data deposition: The NMR assignments and coordinates for Y48pCMF cytochrome c reported in this paper have been deposited in the Biological Magnetic Resonance Data Bank (BMRB entry code 25660) and Protein Data Bank (PDB ID code 2N3Y) databases, respectively.

  • This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1618008114/-/DCSupplemental.

Freely available online through the PNAS open access option.

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