Regulation of Cytochrome C by Phosphorylation: Mitochondrial Respiration and Apoptosis

Mammalian cytochrome c (Cytc) is a small globular protein and functions as a mobile electron carrier in the electron transport chain (ETC), shuttling electrons from bc1 complex to cytochrome c oxidase (COX). In addition, Cytc participates in type II apoptosis, when it is released from the mitochondria. Such a molecule, central to life and death decisions of the cell, should be tightly controlled. We previously showed that Cytc is regulated by cell signaling pathways in a highly tissue-specific manner and that Cytc isolated from heart and liver is phosphorylated on Tyr97 and Tyr48, respectively. Here we add another phosphorylation site and show that Cytc isolated from kidney is phosphorylated on Thr28. This phosphorylation is likely mediated by AMP kinase. Phosphorylated and Thr28Glu phosphomimetic Cytc cause partial inhibition in the reaction with COX supporting our model that under healthy conditions ETC proteins are phosphorylated to limit electron flux in the electron transport chain, which in turn prevents a hyperpolarization of the mitochondrial membrane potential, a known cause of reactive oxygen species (ROS) production and trigger of apoptosis. To test our model we generated Cytc knockout cells expressing Thr28Glu phosphomimetic Cytc. We observed reduction of intact cellular respiration, mitochondrial membrane potential, and ROS. Finally, 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 COX and thus suggests a mechanism for the altered enzyme kinetics.