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