Mammalian liver cytochrome c is tyrosine-48 phosphorylated in vivo, inhibiting mitochondrial respiration

Biochim Biophys Acta. 2008 Jul-Aug;1777(7-8):1066-71. doi: 10.1016/j.bbabio.2008.04.023. Epub 2008 Apr 22.


Cytochrome c (Cyt c) is part of the mitochondrial electron transport chain (ETC), accepting electrons from bc(1) complex and transferring them to cytochrome c oxidase (CcO). The ETC generates the mitochondrial membrane potential, which is used by ATP synthase to produce ATP. In addition, the release of Cyt c from the mitochondria often commits a cell to undergo apoptosis. Considering its central role in life (respiration) and death (apoptosis) decisions one would expect tight regulation of Cyt c function. Reversible phosphorylation is a main cellular regulatory mechanism, but the effect of cell signaling targeting the mitochondrial oxidative phosphorylation system is not well understood, and only a small number of proteins that can be phosphorylated have been identified to date. We have recently shown that Cyt c isolated from cow heart tissue is phosphorylated on tyrosine 97 in vivo, which leads to inhibition of respiration in the reaction with CcO. In this study we isolated Cyt c from a different organ, cow liver, under conditions preserving the physiological phosphorylation state. Western analysis with a phosphotyrosine specific antibody suggested that liver Cyt c is phosphorylated. Surprisingly, the phosphorylation site was unambiguously assigned to Tyr-48 by immobilized metal affinity chromatography/nano-liquid chromatography/electrospray ionization mass spectrometry (IMAC/nano-LC/ESI-MS), and not to the previously identified phospho-Tyr-97 in cow heart. As is true of Tyr-97, Tyr-48 is conserved in eukaryotes. As one possible consequence of Tyr-48 phosphorylation we analyzed the in vitro reaction kinetics with isolated cow liver CcO revealing striking differences. Maximal turnover of Tyr-48 phosphorylated Cyt c was 3.7 s(-1) whereas dephosphorylation resulted in a 2.2 fold increase in activity to 8.2 s(-1). Effects of Tyr-48 phosphorylation based on the Cyt c crystal structure are discussed.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Cattle
  • Chromatography, Affinity
  • Cytochromes c / chemistry
  • Cytochromes c / isolation & purification
  • Cytochromes c / metabolism*
  • Electron Transport Complex IV / chemistry
  • Electron Transport Complex IV / metabolism*
  • Liver / enzymology*
  • Models, Molecular
  • Oxygen Consumption / physiology*
  • Phosphorylation
  • Phosphotyrosine / metabolism*
  • Tyrosine / metabolism


  • Phosphotyrosine
  • Tyrosine
  • Cytochromes c
  • Electron Transport Complex IV