Mimicking Tyrosine Phosphorylation in Human Cytochrome c by the Evolved tRNA Synthetase Technique

Alejandra Guerra-Castellano, Antonio Díaz-Quintana, Blas Moreno-Beltrán, Javier Lõpez-Prados, Pedro M. Nieto, Wiebke Meister, Jana Staffa, Miguel Teixeira, Peter Hildebrandt, Miguel A. De La Rosa, Irene Díaz-Moreno

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25 Citations (Scopus)


Phosphorylation of tyrosine 48 of cytochrome c is related to a wide range of human diseases due to the pleiotropic role of the heme-protein in cell life and death. However, the structural conformation and physicochemical properties of phosphorylated cytochrome c are difficult to study as its yield from cell extracts is very low and its kinase remains unknown. Herein, we report a high-yielding synthesis of a close mimic of phosphorylated cytochrome c, developed by optimization of the synthesis of the non-canonical amino acid p-carboxymethyl-L-phenylalanine (pCMF) and its efficient site-specific incorporation at position 48. It is noteworthy that the Y48pCMF mutation significantly destabilizes the Fe-Met bond in the ferric form of cytochrome c, thereby lowering the pKa value for the alkaline transition of the heme-protein. This finding reveals the differential ability of the phosphomimic protein to drive certain events. This modified cytochrome c might be an important tool to investigate the role of the natural protein following phosphorylation.

Original languageEnglish
Pages (from-to)15004-15012
Number of pages9
JournalChemistry-A European Journal
Issue number42
Publication statusPublished - 1 Oct 2015


  • cytochromes
  • heme proteins
  • NMR spectroscopy
  • phosphorylation
  • protein modifications


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