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J Biol Chem. 2017 Jan 6;292(1):64-79. doi: 10.1074/jbc.M116.744664. Epub 2016 Oct 7.

Phosphorylation of Cytochrome c Threonine 28 Regulates Electron Transport Chain Activity in Kidney: IMPLICATIONS FOR AMP KINASE.

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From the Center for Molecular Medicine and Genetics and.
the Departments of Biochemistry and Molecular Biology.
the Chemistry Department and.
the College of Medicine, Dankook University, Cheonan-si, Chungcheongnam-do 31116, Republic of Korea.
the Center for Free Radical and Antioxidant Health and the Department of Environmental and Occupational Health, University of Pittsburgh, Pittsburgh, Pennsylvania 15219.
the Department of Neurology, University of Miami School of Medicine, Miami, Florida 33136, and.
Emergency Medicine, and.
Pharmaceutical Sciences, Wayne State University, Detroit, Michigan 48201.
the Life Sciences Collaborative Access Team, Northwestern University, Center for Synchrotron Research, Argonne, Illinois 60439.
the MCB Department, Brown University, Providence, Rhode Island 02912.
From the Center for Molecular Medicine and Genetics and


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 isolated from kidneys is phosphorylated on Thr28, leading to a partial 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 superior behavior regarding protein stability and its ability to degrade reactive oxygen species compared with wild-type unphosphorylated Cytc Introduction of T28E phosphomimetic Cytc into Cytc knock-out cells shows that intact cell respiration, mitochondrial membrane potential (ΔΨm), and ROS levels are reduced compared with wild type. As we show by high resolution crystallography of wild-type and T28E Cytc in combination with molecular dynamics simulations, Thr28 is located at a central 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 kidney, 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 ΔΨm hyperpolarization, a known cause of ROS and trigger of apoptosis.


AMP-activated kinase (AMPK); apoptosis; cell signaling; cytochrome c; electron transport chain; electron transport system (ETS); kidney metabolism; mitochondria; oxidative phosphorylation; respiration

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