Cardiac Tyrosine 97 Phosphorylation of Cytochrome c Regulates Respiration and Apoptosis

It was previously reported that tyrosine 97 (Y97) of cytochrome <i>c</i> is phosphorylated in cow heart tissue under physiological conditions. Y97 phosphorylation was shown to partially inhibit respiration in vitro in the reaction with purified cytochrome <i>c</i> oxidase. Here, we use phosphomimetic Y97E Cyt<i>c</i> to further characterize the functional effects of this modification both in vitro and in cell culture models. In vitro, phosphomimetic Y97E Cyt<i>c</i> showed lower activity in the reaction with purified cow heart cytochrome <i>c</i> oxidase (COX), decreased caspase-3 activity, and reduced rate of reduction. Additionally, the phosphomimetic Y97E Cyt<i>c</i> tended to be resistant to heme degradation and showed an increased rate of oxidation. Intact mouse Cyt<i>c</i> double knockout fibroblasts were transfected with plasmids coding for phosphomimetic Y97E Cyt<i>c</i> and other variants. Compared to cells expressing wild-type Cyt<i>c</i>, the cells expressing phosphomimetic Y97E Cyt<i>c</i> showed reduced respiration, mitochondrial membrane potential, and reactive oxygen species production, and protection from apoptosis. In an oxygen-glucose deprivation/reoxygenation cell culture model of ischemia/reperfusion injury, mitochondrial membrane potential and reactive oxygen species production were decreased. These data show that Cyt<i>c</i> phosphorylation controls the overall flux through the electron transport chain by maintaining optimal intermediate ΔΨm potentials for efficient ATP production while minimizing reactive oxygen species production, thus protecting the cell from apoptosis.