Isolation of regulatory-competent, phosphorylated cytochrome C oxidase

Methods Enzymol. 2009;457:193-210. doi: 10.1016/S0076-6879(09)05011-3.


The role of posttranslational modifications, specifically reversible phosphorylation as a regulatory mechanism operating in the mitochondria, is a novel research direction. The mitochondrial oxidative phosphorylation system is a particularly interesting unit because it is responsible for the production of the vast majority of cellular energy in addition to free radicals, two factors that are aberrant in numerous human diseases and that may be influenced by reversible phosphorylation of the oxidative phosphorylation complexes. We here describe a detailed protocol for the isolation of mammalian liver and heart mitochondria and subsequently cytochrome c oxidase (CcO) under conditions maintaining the physiological phosphorylation state. The protocol employs the use of activated vanadate, an unspecific tyrosine phosphatase inhibitor, fluoride, an unspecific serine/threonine phosphatase inhibitor, and EGTA, a calcium chelator to prevent the activation of calcium-dependent protein phosphatases. CcO purified without manipulation of signaling pathways shows strong tyrosine phosphorylation on subunits II and IV, whereas tyrosine phosphorylation of subunit I can be induced by the cAMP- and TNFalpha-dependent pathways in liver. Using our protocol on cow liver tissue we further show the identification of a new phosphorylation site on CcO subunit IV tyrosine 11 of the mature protein (corresponding to tyrosine 33 of the precursor peptide) via immobilized metal affinity chromatography/nano-liquid chromatography/electrospray ionization mass spectrometry (IMAC/nano-LC/ESI-MS). This phosphorylation site is located close to the ATP and ADP binding site, which adjusts CcO activity to cellular energy demand, and we propose that phosphorylation of tyrosine 11 enables allosteric regulation.

MeSH terms

  • Animals
  • Cattle
  • Chromatography, Liquid
  • Electron Transport Complex IV / analysis
  • Electron Transport Complex IV / isolation & purification*
  • Electron Transport Complex IV / metabolism*
  • Mass Spectrometry
  • Mitochondria, Heart / enzymology*
  • Mitochondria, Liver / enzymology*
  • Phosphorylation
  • Protein Subunits / analysis
  • Protein Subunits / metabolism
  • Spectrometry, Mass, Electrospray Ionization
  • Tyrosine / analysis
  • Tyrosine / metabolism
  • Vanadates / metabolism


  • Protein Subunits
  • Vanadates
  • Tyrosine
  • Electron Transport Complex IV