Cytochrome c (cytc) is a multifunctional protein, acting as an electron carrier in the electron transport chain (ETC), where it shuttles electrons from bc1 complex to cytochrome c oxidase (COX), and as a trigger of type II apoptosis when released from the mitochondria. We previously showed that cytc is regulated in a highly tissue-specific manner: Cytc isolated from heart, liver, and kidney is phosphorylated on Y97, Y48, and T28, respectively. Here, we have analyzed the effect of a new Cytc phosphorylation site, threonine 58, which we mapped in rat kidney Cytc by mass spectrometry. We generated and overexpressed wild-type, phosphomimetic T58E, and two controls, T58A and T58I cytc; the latter replacement is found in human and testis-specific Cytc. In vitro, COX activity, caspase-3 activity, and heme degradation in the presence of H2o2 were decreased with phosphomimetic Cytc compared to wild-type. Cytc-knockout cells expressing T58E or T58I Cytc showed a reduction in intact cell respiration, mitochondrial membrane potential (∆Ψm), ROS production, and apoptotic activity compared to wild-type. We propose that, under physiological conditions, Cytc is phosphorylated, which controls mitochondrial respiration and apoptosis. Under conditions of stress Cytc phosphorylations are lost leading to maximal respiration rates, ∆Ψm hyperpolarization, ROS production, and apoptosis.
Biochemistry | Molecular Biology
Wan, J., Kalpage, H.A., Vaishnav, A., Liu, J., Lee, I., Mahapatra, G. Turner, A.A., Zurek, M.P., Ji, Q., Moraes, C.T., Recanati, M.A., Grossman, L.I., Salomon,A.R., Edwards, B.F.P., Hüttemann, M. Regulation of Respiration and Apoptosis by Cytochrome c Threonine 58 Phosphorylation. Scientific Reports 9: 15815 (2019). https://doi.org/10.1038/s41598-019-52101-z