Abstract
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.
Highlights
Introduction ofT28E cytochrome c (Cytc) into Cytc Double Knock-out Cells Reduces Oxygen Consumption Rate, Mitochondrial Membrane Potential, and reactive oxygen species (ROS)—To test the effect of phosphomimetic substitution of Cytc in intact cells on mitochondrial parameters, we generated cell lines stably transfected with empty vector control and WT, T28E, and T28A Cytc, using mouse lung fibroblasts in which both the somatic and testes-specific Cytc isoforms have been knocked out
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
Mammalian Kidney Cytochrome c Is Phosphorylated on Thr28, Leading to “Controlled Respiration”—To test whether Cytc phosphorylation occurs in mammalian tissues other than the heart and liver, where it is tyrosine-phosphorylated, we chose the kidney, an organ with a high mitochondrial capacity and the second highest oxygen consumption rate only after heart [12]
Summary
Cytochrome c (Cytc) is a small (12-kDa) globular nucleusencoded mitochondrial protein containing a covalently attached heme group with multiple functions. Cytc is released from mitochondria into the cytosol, where it interacts with Apaf-1 to form the apoptosome, which in turn activates caspase-9 and the downstream executioner caspase cascade. Under healthy, nonapoptotic conditions, Cytc acts as a scavenger of reactive oxygen species (ROS) [3], and it takes part in other redox reactions inside mitochondria, including redox-coupled protein import [4] and reduction of p66Shc, a protein that is implicated in the generation of ROS and apoptosis [5]. Given the multiple functions of Cytc, it is not surprising that it is tightly regulated.
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