Serine‐47 phosphorylation of cytochrome c in the mammalian brain regulates cytochrome c oxidase and caspase‐3 activity

Cytochrome c (Cyt c ) is a multifunctional protein that operates as an electron carrier in the mitochondrial electron transport chain and plays a key role in apoptosis. We have previously shown that tissue‐specific phosphorylations of Cyt c in the heart, liver, and kidney play an important role in the regulation of cellular respiration and cell death. Here, we report that Cyt c purified from mammalian brain is phosphorylated on S47 and that this phosphorylation is lost during ischemia. We have characterized the functional effects in vitro using phosphorylated Cyt c purified from pig brain tissue and a recombinant phosphomimetic mutant (S47E). We crystallized S47E phosphomimetic Cyt c at 1.55 Å and suggest that it spatially matches S47‐phosphorylated Cyt c , making it a good model system. Both S47‐phosphorylated and phosphomimetic Cyt c showed a lower oxygen consumption rate in reaction with isolated Cyt c oxidase, which we propose maintains intermediate mitochondrial membrane potentials under physiologic conditions, thus minimizing production of reactive oxygen species. S47‐phosphorylated and phosphomimetic Cyt c showed lower caspase‐3 activity. Furthermore, phosphomimetic Cyt c had decreased cardiolipin peroxidase activity and is more stable in the presence of H 2 O 2 . Our data suggest that S47 phosphorylation of Cyt c is tissue protective and promotes cell survival in the brain.—Kalpage, H. A., Vaishnav, A., Liu, J., Varughese, A., Wan, J., Turner, A. A., Ji, Q., Zurek, M. P., Kapralov, A. A., Kagan, V. E., Brunzelle, J. S., Recanati, M.‐A., Grossman, L. I., Sanderson, T. H., Lee, I., Salomon, A. R., Edwards, B. F. P, Hüttemann, M. Serine‐47 phosphorylation of cytochrome c in the mammalian brain regulates cytochrome c oxidase and caspase‐3 activity. FASEB J. 33, 13503‐13514 (2019). www.fasebj.org