­Role of Cytochrome c Phosphorylation in Regulation of Respiration and Apoptosis

Abstract

Cytochrome c is a functionally diverse protein, carrying electrons in the electron transport chain and playing a critical role in cellular apoptosis. Previous work by our lab suggest the importance of reversible phosphorylation of cytochrome c in regulating its functions. We hypothesize that under healthy conditions, phosphorylated cytochrome c partially inhibits mitochondrial respiration and maintains healthy mitochondrial membrane potential, preventing ROS generation, while cellular stress‐mediated dephosphorylation leads to increased respiration and ROS generation, initiating apoptosis.In this study, we purified bovine kidney cytochrome c in the presence of phosphatase inhibitors, identified threonine phosphorylation by immunoblot analysis, and determined threonine 28 phosphorylation by Nano/LC/ESI/MS/MS. To characterize its effect, Thr28 was mutated to glutamate, a phosphomimetic mutation, and alanine, a nonphosphorylatable control, and experiments were performed with bacterially overexpressed proteins. In the reaction with cytochrome c oxidase, Thr28Glu showed significant reduced respiration rates compared to wild‐type, with no alteration in caspase‐3 activation. Thr28Glu had a higher reduction rate from an initial oxidized state, with no change in the rate of oxidation compared to wild‐type, and was found to be resistant to heme destruction in the presence of excess ROS. Our data suggest that Thr28, conserved in mammalian cytochrome c, is an important regulatory site.