Mammalian cytochrome c (Cytc) plays a key role in cellular life and death decisions, functioning as an electron carrier in the electron transport chain and as a trigger of apoptosis when released from the mitochondria. However, its regulation is not well understood. We show that the major fraction of Cytc iso- lated from kidneys is phosphorylated on Thr28, leading to a par- tial inhibition of respiration in the reaction with cytochrome c oxidase. To further study the effect of Cytc phosphorylation in vitro, we generated T28E phosphomimetic Cytc, revealing supe- rior behavior regarding protein stability and its ability to degrade reactive oxygen species compared with wild-type un- phosphorylated Cytc. Introduction of T28E phosphomimetic Cytc into Cytc knock-out cells shows that intact cell respiration, mitochondrial membrane potential (����m), and ROS levels are reduced compared with wild type. As we show by high resolu- tion crystallography of wild-type and T28E Cytc in combination with molecular dynamics simulations, Thr28 is located at a cen- tral position near the heme crevice, the most flexible epitope of the protein apart from the N and C termini. Finally, in silico prediction and our experimental data suggest that AMP kinase, which phosphorylates Cytc on Thr28 in vitro and colocalizes with Cytc to the mitochondrial intermembrane space in the kid- ney, is the most likely candidate to phosphorylate Thr28 in vivo. We conclude that Cytc phosphorylation is mediated in a tissue- specific manner and leads to regulation of electron transport chain flux via “controlled respiration,” preventing ����m hyperpolarization, a known cause of ROS and trigger of apoptosis.
Biochemistry | Molecular Biology
Mahapatra, G., Varughese, A., Ji, Q., Lee, I., Liu, J., Vaishnav, A., Sinkler, C., Kapralov, A.A., Moraes, C.T., Sanderson, T.H., Stemmler, T.L., Grossman, L.I., Kagan, V.E., Brunzelle, J.S., Salomon, A.R., Edwards, B.F.P., Hüttemann, M. Phosphorylation of Cytochrome c Threonine 28 Regulates Electron Transport Chain Activity in Kidney: Implications for AMP Kinase. J. Biol. Chem. 292: 64-79 (2017). https://doi.org/10.1074/jbc.M116.744664