Brain-Specific Serine-47 Modification of Cytochrome c Regulates Cytochrome c Oxidase Activity Attenuating ROS Production and Cell Death: Implications for Ischemia/Reperfusion Injury and Akt Signaling

We previously reported that serine-47 (S47) phosphorylation of cytochromec(Cytc) in the brain results in lower cytochromecoxidase (COX) activity and caspase-3 activity in vitro. We here analyze the effect of S47 modification in fibroblast cell lines stably expressing S47E phosphomimetic Cytc, unphosphorylated WT, or S47A Cytc. Our results show that S47E Cytcresults in partial inhibition of mitochondrial respiration corresponding with lower mitochondrial membrane potentials (Delta psi(m)) and reduced reactive oxygen species (ROS) production. When exposed to an oxygen-glucose deprivation/reoxygenation (OGD/R) model simulating ischemia/reperfusion injury, the CytcS47E phosphomimetic cell line showed minimal ROS generation compared to the unphosphorylated WT Cytccell line that generated high levels of ROS upon reoxygenation. Consequently, the S47E Cytccell line also resulted in significantly lower cell death upon exposure to OGD/R, confirming the cytoprotective role of S47 phosphorylation of Cytc. S47E Cytcalso resulted in lower cell death upon H(2)O(2)treatment. Finally, we propose that pro-survival kinase Akt (protein kinase B) is a likely mediator of the S47 phosphorylation of Cytcin the brain. Akt inhibitor wortmannin abolished S47 phosphorylation of Cytc, while the Akt activator SC79 maintained S47 phosphorylation of Cytc. Overall, our results suggest that loss of S47 phosphorylation of Cytcduring brain ischemia drives reperfusion injury through maximal electron transport chain flux, Delta psi(m)hyperpolarization, and ROS-triggered cell death.