Structural and functional characterization of phosphomimetic mutants of cytochrome c at threonine 28 and serine 47

Abstract

Protein function is frequently modulated by post-translational modifications of specific residues. Cytochrome c, in particular, is phosphorylated in vivo at threonine 28 and serine 47. However, the effect of such modifications on the physiological functions of cytochrome c - namely, the transfer of electrons in the respiratory electron transport chain and the triggering of programmed cell death - is still unknown. Here we replace each of these two residues by aspartate, in order to mimic phosphorylation, and report the structural and functional changes in the resulting cytochrome c variants. We find that the T28D mutant causes a 30-mV decrease on the midpoint redox potential and lowers the affinity for the distal site of Arabidopsis thaliana cytochrome c1 in complex III. Both the T28D and S47D variants display a higher efficiency as electron donors for the cytochrome c oxidase activity of complex IV. In both protein mutants, the peroxidase activity is significantly higher, which is related to the ability of cytochrome c to leave the mitochondria and reach the cytoplasm. We also find that both mutations at serine 47 (S47D and S47A) impair the ability of cytoplasmic cytochrome c to activate the caspases cascade, which is essential for triggering programmed cell death.

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

“…6D). However, the presence of DOPC:TOCL vesicles, at a 1:100 Cc:lipid ratio, increased the enzymatic activity of both WT and Y48pCMF Cc, similar to that observed for other phosphomimetic Cc mutants (70). Note that the slightly lower peroxidase activity increment observed for Y48pCMF Cc is likely due to its higher population of free protein compared with WT Cc (Fig.…”

“…Notably, the presence of free hemeprotein at high lipid concentrations suggested that Y48pCMF Cc has a lower affinity than WT toward DOPC:TOCL liposomes (Fig. 6B), as recently observed for the phosphomimetic mutant S47D Cc (70). Another interesting finding is the unspecific interaction of Cc-either the WT or Y48pCMF species-with DOPC vesicles (Fig.…”

Phosphorylation of Cytochrome c Threonine 28 Regulates Electron Transport Chain Activity in Kidney

“…This may also at least in part account for the finding of lower Cytc protein levels in cells expressing this mutant. Another evolutionarily forbidden substitution, T28D, which introduces a negative charge, also generates "rogue" functional changes in Cytc, as seen in the reaction with CcO, that are even opposite (29) of what we report for in vivo phosphorylated Cytc, whereas glutamate replacement, as used here, produces the same functional effects as phosphorylated Cytc.…”

Tissue‐specific regulation of cytochrome c by post‐translational modifications: respiration, the mitochondrial membrane potential, ROS, and apoptosis

“…Structural analysis of Cytc has shown that the residues present in this turn have the highest root-mean-square deviations of the whole Cytc molecule, suggesting that this is the most flexible element of the protein (76). However, Thr28 phosphorylation does not have an impact on apoptosome activity, because Thr28 is not part of the Apaf-1 binding domain of Cytc (76,77). It would be interesting to analyze whether Cytc in skeletal muscle is targeted by AMPK (74, 75) or a different kinase.…”