Tyrosine 67 Phosphorylation Controls Respiration and Limits the Apoptotic Functions of Cytochrome c

Cytochrome <i>c</i> (Cyt<i>c</i>) is a multifunctional protein, essential for respiration and intrinsic apoptosis. Post-translational modifications of Cyt<i>c</i> have been linked to physiological and pathophysiologic conditions, including cancer. Cyt<i>c</i> tyrosine 67 (Y67) is a conserved residue that is important to the structure and function of Cyt<i>c.</i> We here report the phosphorylation of Y67 of Cyt<i>c</i> purified from bovine heart mapped by mass spectrometry. We characterized the functional effects of Y67 Cyt<i>c</i> modification using in vitro and cell culture models. Y67 was mutated to the phosphomimetic glutamate (Y67E) and to phenylalanyl (Y67F) as a control. The phosphomimetic Y67E Cyt<i>c</i> inhibited cytochrome <i>c</i> oxidase (COX) activity, redirecting energy metabolism toward glycolysis, and decreased the pro-apoptotic capabilities of Cyt<i>c</i>. The phosphomimetic Y67E Cyt<i>c</i> showed a significantly impaired rate of superoxide scavenging and a reduced rate of oxidation by hydrogen peroxide<i>,</i> suggesting a lower ability to transfer electrons and scavenge reactive oxygen species (ROS). Phosphomimetic Y67E replacement led to an almost complete loss of cardiolipin peroxidase activity, pointing to a central role of Y67 for this catalytic function of Cyt<i>c</i>. In intact cells, phosphomimetic replacement leads to a reduction in cell respiration, mitochondrial membrane potential, and ROS levels. We propose that Y67 phosphorylation is cardioprotective and promotes cell survival.