Superoxide activates mitochondrial uncoupling proteins


Uncoupling protein 1 (UCP1) diverts energy from ATP synthesis to thermogenesis in the mitochondria of brown adipose tissue by catalysing a regulated leak of protons across the inner membrane1,2. The functions of its homologues, UCP2 and UCP3, in other tissues are debated3,4. UCP2 and UCP3 are present at much lower abundance than UCP1, and the uncoupling with which they are associated is not significantly thermogenic5,6. Mild uncoupling would, however, decrease the mitochondrial production of reactive oxygen species, which are important mediators of oxidative damage7,8. Here we show that superoxide increases mitochondrial proton conductance through effects on UCP1, UCP2 and UCP3. Superoxide-induced uncoupling requires fatty acids and is inhibited by purine nucleotides. It correlates with the tissue expression of UCPs, appears in mitochondria from yeast expressing UCP1, and is absent in skeletal muscle mitochondria from UCP3 knockout mice. Our findings indicate that the interaction of superoxide with UCPs may be a mechanism for decreasing the concentrations of reactive oxygen species inside mitochondria.

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Figure 1: Effect of superoxide on the proton conductance of skeletal muscle mitochondria: superoxide activation of UCP3.
Figure 2: Effect of superoxide on the proton conductance of mitochondria from different tissues: superoxide activation of UCP2.
Figure 3: Effect of superoxide on the proton conductance of mitochondria from brown adipose tissue and transgenic yeast: superoxide activation of UCP1.
Figure 4: Nucleotide specificity and affinity of UCP2 (kidney) and UCP3 (skeletal muscle).


  1. 1

    Nicholls, D. G. & Rial, E. A history of the first uncoupling protein, UCP1. J. Bioenerg. Biomembr. 31, 399–406 (1999).

  2. 2

    Klingenberg, M. & Echtay, K. S. Uncoupling proteins: the issues from a biochemist point of view. Biochim. Biophys. Acta 1504, 128–143 (2001).

  3. 3

    Stuart, J. A., Cadenas, S., Jekabsons, M. B., Roussel, D. & Brand, M. D. Mitochondrial proton leak and the uncoupling protein 1 homologues. Biochim. Biophys. Acta 1504, 144–158 (2001).

  4. 4

    Ricquier, D. & Bouillaud, F. The uncoupling protein homologues: UCP1, UCP2, UCP3, StUCP and AtUCP. Biochem. J. 345, 161–179 (2000).

  5. 5

    Arsenijevic, D. et al. Disruption of the uncoupling protein-2 gene in mice reveals a role in immunity and reactive oxygen species production. Nature Genet. 26, 435–439 (2000).

  6. 6

    Vidal-Puig, A. J. et al. Energy metabolism in uncoupling protein 3 gene knockout mice. J. Biol. Chem. 275, 16258–16266 (2000).

  7. 7

    Papa, S. & Skulachev, V. P. Reactive oxygen species, mitochondria, apoptosis and aging. Mol. Cell Biochem. 174, 305–319 (1997).

  8. 8

    Brand, M. D. Uncoupling to survive? The role of mitochondrial inefficiency in ageing. Exp. Gerontol. 35, 811–820 (2000).

  9. 9

    Echtay, K. S., Winkler, E. & Klingenberg, M. Coenzyme Q is an obligatory cofactor for uncoupling protein function. Nature 408, 609–613 (2000).

  10. 10

    Echtay, K. S., Winkler, E., Frischmuth, K. & Klingenberg, M. Uncoupling proteins 2 and 3 are highly active H+ transporters and highly nucleotide sensitive when activated by coenzyme Q (ubiquinone). Proc. Natl Acad. Sci. USA 98, 1416–1421 (2001).

  11. 11

    Echtay, K. S. & Brand, M. D. Coenzyme Q induces GDP sensitive proton conductance in kidney mitochondria. Biochem. Soc. Trans. 29, 763–768 (2001).

  12. 12

    Cadenas, S. et al. UCP2 and UCP3 rise in starved rat skeletal muscle but mitochondrial proton conductance is unchanged. FEBS Lett. 462, 257–260 (1999).

  13. 13

    Pecqueur, C. et al. Uncoupling Protein 2, in vivo distribution, induction upon oxidative stress, and evidence for translational regulation. J. Biol. Chem. 276, 8705–8712 (2001).

  14. 14

    Ruch, W., Cooper, P. H. & Baggiolini, M. Assay of H2O2 production by macrophages and neutrophils with homovanillic acid and horse-radish peroxidase. J. Immunol. Methods 63, 347–357 (1983).

  15. 15

    Zhang, C. Y. et al. Uncoupling protein-2 negatively regulates insulin secretion and is a major link between obesity, β-cell dysfunction and type 2 diabetes. Cell 105, 745–755 (2001).

  16. 16

    Bathgate, B., Freebairn, E. M., Greenland, A. J. & Reid, G. A. Functional expression of the rat brown adipose tissue uncoupling protein in Saccharomyces cerevisiae. Mol. Microbiol. 6, 363–370 (1992).

  17. 17

    Echtay, K. S., Bienengraeber, M. & Klingenberg, M. Mutagenesis of the uncoupling protein of brown adipose tissue. Neutralization of E190 largely abolishes pH control of nucleotide binding. Biochemistry 36, 8253–8260 (1997).

  18. 18

    Stuart, J. A., Harper, J. A., Jekabsons, M. B., Brindle, K. M. & Brand, M. D. A mitochondrial uncoupling artefact can be caused by expression of uncoupling protein 1 in yeast. Biochem. J. 356, 779–789 (2001).

  19. 19

    Cadenas, S. et al. AMP decreases the efficiency of skeletal-muscle mitochondria. Biochem. J. 351, 307–311 (2000).

  20. 20

    Halliwell, B. & Gutteridge, J. M. C. Free Radicals in Biology and Medicine 2nd edn (Clarendon, Oxford, 1989).

  21. 21

    Pastore, D., Fratianni, A., Di Pede, S. & Passarella, S. Effects of fatty acids, nucleotides and reactive oxygen species on durum wheat mitochondria. FEBS Lett. 470, 88–92 (2000).

  22. 22

    Kowaltowski, A. J., Costa, A. D & Vercesi, A. E. Activation of the potato plant uncoupling mitochondrial protein inhibits reactive oxygen species generation by the respiratory chain. FEBS Lett. 425, 213–216 (1998).

  23. 23

    Jezek, P., Orosz, D. E., Modriansky, M. & Garlid, K. D. Transport of anions and protons by the mitochondrial uncoupling protein and its regulation by nucleotides and fatty acids. A new look at old hypotheses. J. Biol. Chem. 269, 26184–26190 (1994).

  24. 24

    Liu, S. S. Generating, partioning, targeting and functioning of superoxide in mitochondria. Biosci. Rep. 17, 259–272 (1997).

  25. 25

    Laloi, M. et al. A plant cold-induced uncoupling protein. Nature 389, 135–136 (1997).

  26. 26

    Scandalios, J. G. Oxygen stress and superoxide dismutases. Plant Physiol. 101, 7–12 (1993).

  27. 27

    Negre-Salvayre, A. et al. A role for uncoupling protein-2 as a regulator of mitochondrial hydrogen peroxide generation. FASEB J. 11, 809–15 (1997).

  28. 28

    Brand, M. D. in Bioenergetics—a Practical Approach (eds Brown, G. C. & Cooper, C. E.) 39–62 (IRL, Oxford, 1995).

  29. 29

    Rolfe, D. F. S., Hulbert, A. J. & Brand, M. D. Characteristics of mitochondrial proton leak and control of oxidative phosphorylation in the major oxygen-consuming tissues of the rat. Biochim. Biophys. Acta 1188, 405–16 (1994).

  30. 30

    Miyazaki, J. et al. Establishment of a pancreatic beta cell line that retains glucose-inducible insulin secretion: special reference to expression of glucose transporter isoforms. Endocrinology 127, 126–132 (1990).

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We thank A. Abuin for help in constructing the UCP3 knockout mice.

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Correspondence to Martin D. Brand.

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