Phosphorylation of cytochrome C: functional and structural features

Abstract

Post-translational modifications often modulate protein function. Actually, phosphorylation of cytochrome c occurs in vivo at threonine 28, serine 47 and tyrosines 48 and 97. Phosphorylation of the two latters, in particular, is related to a wide range of human diseases as cytochrome c plays a pleiotropic role serving as an electron carrier in the respiratory electron transfer and acting as a cell death signal at the onset of apoptosis. The effect that phosphorylation of threonine 28 and serine 47 bears on the physiological functions of this protein remains concealed. The low yield of phosphorylated cytochrome c purification from cell extracts makes its analysis challenging. Also the specific kinases acting on the protein remain unknown. Hence, it has resorted to mutations to mimic targeted phosphorylation. Here, we have replaced threonine 28 and serine 47 by aspartate. And the analysis of tyrosine 48 and 97 phosphorylation has been performed by using the non-canonical amino acid p-carboxymethyl-L-phenylalanine (pCMF), which is a close phosphorylation mimic of tyrosine. Noteworthy, the Y48pCMF mutation significantly lowers the value for the alkaline transition pKa of oxidized cytochrome c. The negative charges at positions 28 and 48 cause a decrease in the midpoint redox potential value of 30 mV and 60 mV, respectively, and lower the affinity towards the distal site of cytochrome c1 in complex III. However, the phosphomimic variants at position 28, 47 and 48 are more efficient as electron donors to cytochrome c oxidase than the wild-type species. Concerning the role of cytochrome c in programmed cell death, negative charges at positions 48 and 97 hinder its ability to triger caspase-3 activation. In addition, any modification of residue 47 affects the pro-apoptotic function of cytochrome c. In summary, phosphorylation of cytochrome c modulates its distinct functions depending on the targeted residue, and can thus be the basis to understand an ample set of molecular diseases.