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

Abstract

Regulation of mitochondrial activity allows cells to adapt to changing conditions and to control oxidative stress, and its dysfunction can lead to hypoxia-dependent pathologies such as ischemia and cancer. Although cytochrome c phosphorylation—in particular, at tyrosine 48—is a key modulator of mitochondrial signaling, its action and molecular basis remain unknown. Here we mimic phosphorylation of cytochrome c by replacing tyrosine 48 with p-carboxy-methyl-l-phenylalanine (pCMF). The NMR structure of the resulting mutant reveals significant conformational shifts and enhanced dynamics around pCMF that could explain changes observed in its functionality: The phosphomimetic mutation impairs cytochrome c diffusion between respiratory complexes, enhances hemeprotein peroxidase and reactive oxygen species scavenging activities, and hinders caspase-dependent apoptosis. Our findings provide a framework to further investigate the modulation of mitochondrial activity by phosphorylated cytochrome c and to develop novel therapeutic approaches based on its prosurvival effects.

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

“…However, the situation is different with regard to apoptosis, where some PTMs seem to affect apoptosome formation and downstream caspase activation, whereas others do not. It is noteworthy that Cytc Tyr48 phosphorylation fully protects against caspase activation, as shown with phosphomimetic Cytc (64,66), whereas Thr28 phosphorylation in kidney has no such effect. Liver cells are constantly bombarded with molecules that the organism takes up through the digestive system and even the lung (91).…”

“…The phosphomimetic Tyr48Glu Cytc demonstrated significant effects on the apoptotic function of Cytc by decreasing cardiolipin peroxidase activity and, strikingly, by abolishing downstream caspase-3 activation (66). A study using the artificial phosphomimetic amino acid, p-carboxymethyl phenylalanine, substituted for Tyr48, showed an ;70% reduction in caspase 3 activity, whereas cardiolipin peroxidase activity did not change when data were normalized for baseline peroxidase activity in the absence of cardiolipin (64). This study also concluded that Tyr48pCMF Cytc reduces ETC activity compared to WT protein.…”

“…2). It is also important to note that Tyr48 is located in the 40-57 V loop, which requires the least free energy to be unfolded (63,64). Both Tyr48 and Tyr97 residues showed NMR signal intensity changes after cardiolipin binding, whereas Tyr67 and Tyr74 did not, suggesting that changes in Tyr48 and Tyr97 more strongly affect the molecular dynamics of Cytc (65).…”

Mitochondrial cytochromecliberates the nucleophosmin-sequestered ARF tumor suppressor in the nucleolus

“…Most shifted signals mapped to fringes of the heme cleft ( Fig. 3b), as in other interactions with Cc partners (14,33,34). Four lysine residues located at the interface.…”

Structural Biology of Iron‐Binding Proteins by NMR Spectroscopy

“…The effects induced by mutation of Gly41 on the structural and dynamic properties of cyt c have been the subject of NMR activities by K. Bren and co‐workers , . Instead, we have collaborated with the group of M. de La Rosa for the NMR characterization of the phosphomimetic variant of human cyt c obtained by replacing Tyr48 by the synthetic amino‐acid p‐carboxy‐methyl‐ l ‐phenylalanine (pCMF) . The site‐specific incorporation of pCMF at position 48 by the evolved tRNA synthetase technique was required due to the difficulties in obtaining the phosphorylated protein because its yield from cell extracts is low and the phosphorylating kinase is not identified.…”