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. 2011 Jun 8;13(6):712-9.
doi: 10.1016/j.cmet.2011.03.024.

Protein phosphorylation and prevention of cytochrome oxidase inhibition by ATP: coupled mechanisms of energy metabolism regulation

Rebeca Acin-Perez  1 , Domenico L Gatti, Yidong Bai, Giovanni Manfredi
Affiliations

Protein phosphorylation and prevention of cytochrome oxidase inhibition by ATP: coupled mechanisms of energy metabolism regulation

Rebeca Acin-Perez et al. Cell Metab. .

Abstract

Rapid regulation of oxidative phosphorylation is crucial for mitochondrial adaptation to swift changes in fuels availability and energy demands. An intramitochondrial signaling pathway regulates cytochrome oxidase (COX), the terminal enzyme of the respiratory chain, through reversible phosphorylation. We find that PKA-mediated phosphorylation of a COX subunit dictates mammalian mitochondrial energy fluxes and identify the specific residue (S58) of COX subunit IV-1 (COXIV-1) that is involved in this mechanism of metabolic regulation. Using protein mutagenesis, molecular dynamics simulations, and induced fit docking, we show that mitochondrial energy metabolism regulation by phosphorylation of COXIV-1 is coupled with prevention of COX allosteric inhibition by ATP. This regulatory mechanism is essential for efficient oxidative metabolism and cell survival. We propose that S58 COXIV-1 phosphorylation has evolved as a metabolic switch that allows mammalian mitochondria to rapidly toggle between energy utilization and energy storage.

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Figures

Figure 1
Figure 1. C1 cells expressing WT and mutant COXIV-1
A) Western Blot of C1 cells expressing WT, S58A, and S58D COXIV-1 using anti COXIV anitbodies. FpSDH is a loading control. B) First dimension BNGE to detect COX assembly using anti-COXI antibodies. βATPase is a loading control. C) COX activity measured spectrophotometrically in C1 cells expressing COXIV-1. D) Intact cell respiration upon PKA activation or inhibition by 8Br cAMP (1mM, for 2 hrs) or H89 (1μM, for 2 hrs), respectively. E) Modulation assessed by TMPD/ascorbate driven respiration. Bars are averages of mass cultures and 2 clones (in triplicate). Statistical differences are relative to mock C1 cells (transfected with empty vector). A9 mock cells are included as reference. *, p<0.01; ***, p<0.0001. Unt, untransfected cells. F) 2D-BNGE and Western Blot of mitochondria from transfected cells treated with 8Br cAMP (1mM, for 2 hrs), using an anti-phospho Ser/Thr antibody. The dashed box indicates the region of COXIV migration (inset below each panel), when the membrane was re-probed with anti-COXIV antibody. P-COXIV/COXIV ratios are shown (average of three independent gels ± SD). G) Immunoprecipitation of phosphorylated COXIV-1 in mitochondrial lysates from A9 cells with S58phospho-COXIV-1 (Ab6496 or Ab6497) and detection with COXIV antibodies. Rabbit IgG were used as negative control; IP, immunoprecipitation; SN, supernatant. H) Immunoprecipitation of COXIV from untreated and H89 treated (1μM, for 2 hrs) cells expressing WT, S58A, and S58D COXIV-1.
Fig. 2
Fig. 2. Structural models of COX and ATP binding site on the matrix side of COX
A) Molecular surface of membrane (teal) embedded dimeric WT COX. The enzyme is in yellow with the two COXIV subunits in magenta. B) Superposition of the matrix domain of COXIV in WT (blue), S58A (red), S58D (green), and S58phopsho (yellow), as derived from the most prevalent conformations in the MD. C) Binding poses of ATP on the surface of WT COXIV-1 in the most prevalent conformation (Cluster 1). S56 and S58 side chains atoms are color-coded (carbon, grey; oxygen, red; hydrogen, white). D,E) Binding poses of ATP on the surface of WT COXIV-1 in the conformations of Cluster 2 and 3, respectively. F) Binding poses of ATP on the surface of S58phospho COXIV-1 derived from the entire MD simulation superimposed on the conformation of Cluster 1. The phosphate moiety of S58phospho is color-coded (phosphorus, green; carbon, grey; oxygen, red; hydrogen, white). G, H) Molecular surfaces of WT COXIV-1 and S58phospho COXIV-1 colored according to electrostatic potentials (+1 kT/e, blue; −1 kT/e, red). I) Matrix side of COX (yellow surface) embedded in the membrane (shown as sticks) with ATP bound to subunit IV-1 (grey surface).
Figure 3
Figure 3. COX allosteric inhibition by ATP in cells expressing COXIV-1 and its role in OXPHOS
Oxygen consumption using increasing concentrations of cytochrome c in the presence of ATP (5mM) or ADP (5mM) measured in WT (A), S58A (B), and S58D (C) permeabilized COXIV-1 expressing cells (n=3). D) Percentage of ATP inhibition at different cytochrome c concentrations from values from A-C (n=3). E) COX activity measured spectrophotometrically in sonicated mitochondria after pyrophosphatase (PP) treatment (n=6 for each point). F) Doubling time ratio (n=3) of cells grown in glucose versus galactose medium (DT Glu/Gal). Statistically significant differences are relative to WT COXIV-1 cells. *, p<0.01; **, p<0.001; ***, p<0.0001.
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