Phosphomimetic Substitution of CytochromecTyrosine 48 Decreases Respiration and Binding to Cardiolipin and Abolishes Ability to Trigger Downstream Caspase Activation

Abstract

Mammalian cytochrome c (Cytc) transfers electrons from the bc(1) complex to cytochrome c oxidase (CcO) as part of the mitochondrial electron transport chain, and it also participates in type II apoptosis. Our recent discovery of two tyrosine phosphorylation sites in Cytc, Tyr97 in bovine heart and Tyr48 in bovine liver, indicates that Cytc functions are regulated through cell signaling. To characterize the role of Cytc tyrosine phosphorylation in detail using an independent approach, we here overexpressed and purified a Tyr48Glu mutant Cytc, mimicking the in vivo Tyr48 phosphorylation found in cow liver, along with wild-type and Tyr48Phe variants as controls. The midpoint redox potential of the phosphomimetic mutant was decreased by 45 mV compared to control (192 vs 237 mV). Similar to Tyr48 in vivo phosphorylated Cytc, direct kinetic analysis of the Cytc reaction with isolated CcO revealed decreased V(max) for the Tyr48Glu mutant by 30% compared to wild type or the Tyr48Phe variants. Moreover, the phosphomimetic substitution resulted in major changes of Cytc functions related to apoptosis. The binding affinity of Tyr48Glu Cytc to cardiolipin was decreased by about 30% compared to wild type or the Tyr48Phe variants, and Cytc peroxidase activity of the Tyr48Glu mutant was cardiolipin-inducible only at high cardiolipin concentration, unlike controls. Importantly, the Tyr48Glu Cytc failed to induce any detectable downstream activation of caspase-3. Our data suggest that in vivo Tyr48 phosphorylation might serve as an antiapoptotic switch and highlight the strategic position and role of the conserved Cytc residue Tyr48 in regulating multiple functions of Cytc.

Cardiac mitochondrial matrix and respiratory complex protein phosphorylation

“…In both cases, changes in the kinetics of complex IV were observed when using the (at least partially) phosphorylated cytochrome c, which were reverted by alkaline phosphatase treatment. With the Tyr49 modified cytochrome c purified from liver, a twofold decrease in the maximal rate of isolated complex IV was observed (229), similar to what was observed when mutating this residue to Glu to mimic phosphorylation (159). This Glu49 cytochrome c also showed a lower redox potential by 45 mV, had a 30% lower binding affinity to cardiolipin, and was unable to catalyze cardiolipin oxidation or induce caspase-3 activity, which are events linked to the proapoptotic role of cytochrome c when released from the inner mitochondrial membrane (159).…”

“…With the Tyr49 modified cytochrome c purified from liver, a twofold decrease in the maximal rate of isolated complex IV was observed (229), similar to what was observed when mutating this residue to Glu to mimic phosphorylation (159). This Glu49 cytochrome c also showed a lower redox potential by 45 mV, had a 30% lower binding affinity to cardiolipin, and was unable to catalyze cardiolipin oxidation or induce caspase-3 activity, which are events linked to the proapoptotic role of cytochrome c when released from the inner mitochondrial membrane (159). The Tyr98-phosphorylated cytochrome c from heart generated sigmoidal kinetics in isolated complex IV and exhibited a marked blue shift of the 695-nm spectral peak that reflects the interaction of the c heme with surrounding Met residues (119), also suggesting that a high fraction of cytochrome c molecules in the preparation contained this modification.…”

“…This requirement is often overlooked when using high-sensitivity methods for detecting phosphorylated proteins, including phosphopeptide enhancement mass spectrometry (MS) (24,58,121,165,236), radiolabeling (6,29,128), and high-sensitivity MS (156,175), all of which permit the detection of a few copies of phosphorylated peptides in the enzyme protein pool. Another line of evidence for the regulatory role of a protein phosphorylation site may include changes in enzyme activity upon specific amino acid substitution that simulate or eliminate the PTM (1,101,159). Though this might be a powerful approach in some circumstances, the substitution frequently leads to false-positive results because of changes in protein structure or assembly induced by the amino acid substitution that are seductive but could alter activity in a manner that does not actually occur upon phosphorylation of the wild-type residue.…”

Specific nitration of tyrosines 46 and 48 makes cytochrome c assemble a non‐functional apoptosome

“…It has recently been reported that tyrosine 48 gets phosphorylated under homeostatic conditions [37,38], with the concomitant effect on the availability of Cc to activate caspases [39,40]. Nitration and phosphorylation of Cc at the same tyrosine residue are mutually exclusive [41] but inhibit Cc-dependent caspases activation with a similar efficiency.…”

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

“…impair both the electron donation to CcO and the activation of caspase-9 mediated by Apaf-1 in vitro (26,27). Notably, nitration of this residue also hinders the ability of Cc to activate Apaf-1 (28)(29)(30).…”