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

Cytochrome (Cyt) is a multifunctional protein, essential for respiration and intrinsic apoptosis. Post-translational modifications of Cyt have been linked to physiological and pathophysiologic conditions, including cancer. Cyt tyrosine 67 (Y67) is a conserved residue that is important to the structure and function of Cyt We here report the phosphorylation of Y67 of Cyt purified from bovine heart mapped by mass spectrometry. We characterized the functional effects of Y67 Cyt 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 inhibited cytochrome oxidase (COX) activity, redirecting energy metabolism toward glycolysis, and decreased the pro-apoptotic capabilities of Cyt. The phosphomimetic Y67E Cyt showed a significantly impaired rate of superoxide scavenging and a reduced rate of oxidation by hydrogen peroxide 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. 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.