Control of Electron Transport Chain Flux by Cytochrome c Threonine 28 phosphorylation in Kidney: Implications for AMP Kinase

Abstract

Mammalian cytochrome c (Cytc) transfers electrons in the electron transport chain (ETC) but also executes apoptosis and should thus be tightly regulated. We recently showed that heart and liver Cytc is tyrosine phosphorylated. Here we show that Cytc from kidney is phosphorylated on Thr28, likely mediated by AMP kinase, indicating tissue-specific adaptations in metabolic control. In vivo phosphorylated and Thr28Glu phosphomimetic Cytc cause partial inhibition in the reaction with Cytc oxidase, thus helping to control respiration. In Cytc knockout cells expressing Thr28Glu phosphomimetic Cytc we found reduction of intact cellular respiration, mitochondrial membrane potential (DYm), and ROS. High resolution crystal structure analyses and molecular dynamics simulations suggest that the Thr28-containing epitope is the most flexible internal part of Cytc, which is in close proximity to key residues on Cytc oxidase and thus suggests a mechanism for the altered enzyme kinetics. We proposes that under healthy conditions Cytc is phosphorylated to partially inhibit ETC electron flux, preventing DYm hyperpolarization, a known cause of ROS production and trigger of apoptosis. Our data suggest, for the first time, that ETC flux can be controlled at the level of Cytc, making it an attractive target for therapeutic intervention in conditions of mitochondrial dysfunction.