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Affinity gradients drive copper to cellular destinations


Copper is an essential trace element for eukaryotes and most prokaryotes1. However, intracellular free copper must be strictly limited because of its toxic side effects. Complex systems for copper trafficking evolved to satisfy cellular requirements while minimizing toxicity2. The factors driving the copper transfer between protein partners along cellular copper routes are, however, not fully rationalized. Until now, inconsistent, scattered and incomparable data on the copper-binding affinities of copper proteins have been reported. Here we determine, through a unified electrospray ionization mass spectrometry (ESI-MS)-based strategy, in an environment that mimics the cellular redox milieu, the apparent Cu(I)-binding affinities for a representative set of intracellular copper proteins involved in enzymatic redox catalysis, in copper trafficking to and within various cellular compartments, and in copper storage. The resulting thermodynamic data show that copper is drawn to the enzymes that require it by passing from one copper protein site to another, exploiting gradients of increasing copper-binding affinity. This result complements the finding that fast copper-transfer pathways require metal-mediated protein–protein interactions and therefore protein–protein specific recognition3. Together with Cu,Zn-SOD1, metallothioneins have the highest affinity for copper(I), and may play special roles in the regulation of cellular copper distribution; however, for kinetic reasons they cannot demetallate copper enzymes. Our study provides the thermodynamic basis for the kinetic processes that lead to the distribution of cellular copper.

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Figure 1: Determination of the relative Cu(I)-binding affinity of GSH and DETC.
Figure 2: Demetallation of Cu 1 Sco1 and the Cu A site of CcO by apo-MT-2 as followed by ESI-MS.
Figure 3: Free-energy gradients of cellular Cu(I) delivery pathways.


  1. Bertini, I., Cavallaro, G. & McGreevy, K. Cellular copper management – a user’s guide. Coord. Chem. Rev. 254, 506–524 (2010)

    CAS  Article  Google Scholar 

  2. Kim, B. E., Nevitt, T. & Thiele, D. J. Mechanisms for copper acquisition, distribution and regulation. Nature Chem. Biol. 4, 176–185 (2008)

    CAS  Article  Google Scholar 

  3. Banci, L. et al. The Atx1-Ccc2 complex is a metal-mediated protein–protein interaction. Nature Chem. Biol. 2, 367–368 (2006)

    ADS  CAS  Article  Google Scholar 

  4. Rae, T. D., Schmidt, P. J., Pufahl, R. A., Culotta, V. C. & O’Halloran, T. V. Undetectable intracellular free copper: the requirement of a copper chaperone for superoxide dismutase. Science 284, 805–808 (1999)

    ADS  CAS  Article  Google Scholar 

  5. Finney, L. A. & O’Halloran, T. V. Transition metal speciation in the cell: insights from the chemistry of metal ion receptors. Science 300, 931–936 (2003)

    ADS  CAS  Article  Google Scholar 

  6. Nose, Y., Rees, E. M. & Thiele, D. J. Structure of the Ctr1 copper trans‘PORE’ter reveals novel architecture. Trends Biochem. Sci. 31, 604–607 (2006)

    CAS  Article  Google Scholar 

  7. Pufahl, R. A. et al. Metal ion chaperone function of the soluble Cu(I) receptor Atx1. Science 278, 853–856 (1997)

    ADS  CAS  Article  Google Scholar 

  8. Huffman, D. L. & O’Halloran, T. V. Function, structure, and mechanism of intracellular copper trafficking proteins. Annu. Rev. Biochem. 70, 677–701 (2001)

    CAS  Article  Google Scholar 

  9. Linz, R. & Lutsenko, S. Copper-transporting ATPases ATP7A and ATP7B: cousins, not twins. J. Bioenerg. Biomembr. 39, 403–407 (2007)

    CAS  Article  Google Scholar 

  10. Voskoboinik, I. & Camakaris, J. Menkes copper-translocating P-type ATPase(ATP7A): biochemical and cell biology properties, and role in Menkes disease. J. Bioenerg. Biomembr. 34, 363–371 (2002)

    CAS  Article  Google Scholar 

  11. Atkinson, A. & Winge, D. R. Metal acquisition and availability in the mitochondria. Chem. Rev. 109, 4708–4721 (2009)

    CAS  Article  Google Scholar 

  12. Kagi, J. H. R. Metallothionein. Birkhäuser Verlag (1993)

    Google Scholar 

  13. Krezel, A. et al. Coordination of heavy metals by dithiothreitol, a commonly used thiol group protectant. J. Inorg. Biochem. 84, 77–88 (2001)

    CAS  Article  Google Scholar 

  14. Ostergaard, H., Tachibana, C. & Winther, J. R. Monitoring disulfide bond formation in the eukaryotic cytosol. J. Cell Biol. 166, 337–345 (2004)

    CAS  Article  Google Scholar 

  15. Nielson, K. B. & Winge, D. R. Preferential binding of copper to the beta domain of metallothionein. J. Biol. Chem. 259, 4941–4946 (1984)

    CAS  PubMed  Google Scholar 

  16. Banci, L. et al. An NMR study of the interaction between the human copper(I) chaperone and the second and fifth metal-binding domains of the Menkes protein. FEBS J. 272, 865–871 (2005)

    CAS  Article  Google Scholar 

  17. Banci, L. et al. Solution structure and intermolecular interactions of the third metal-binding domain of ATP7A, the Menkes disease protein. J. Biol. Chem. 281, 29141–29147 (2006)

    CAS  Article  Google Scholar 

  18. Banci, L. et al. Human Sco1 functional studies and pathological implications of the P174L mutant. Proc. Natl Acad. Sci. USA 104, 15–20 (2007)

    ADS  CAS  Article  Google Scholar 

  19. Banci, L. et al. Mitochondrial copper(I) transfer from Cox17 to Sco1 is coupled to electron transfer. Proc. Natl Acad. Sci. USA 105, 6803–6808 (2008)

    ADS  CAS  Article  Google Scholar 

  20. Rae, T. D., Torres, A. S., Pufahl, R. A. & O’Halloran, T. V. Mechanism of Cu,Zn-superoxide dismutase activation by the human metallochaperone hCCS. J. Biol. Chem. 276, 5166–5176 (2001)

    CAS  Article  Google Scholar 

  21. Tottey, S. et al. Protein-folding location can regulate manganese-binding versus copper- or zinc-binding. Nature 455, 1138–1142 (2008)

    ADS  CAS  Article  Google Scholar 

  22. Yatsunyk, L. A. & Rosenzweig, A. C. Copper(I) binding and transfer by the N-terminus of the Wilson disease protein. J. Biol. Chem. 282, 8622–8631 (2007)

    CAS  Article  Google Scholar 

  23. Jensen, P. Y., Bonander, N., Moller, L. B. & Farver, O. Cooperative binding of copper(I) to the metal binding domains in Menkes disease protein. Biochim. Biophys. Acta 1434, 103–113 (1999)

    CAS  Article  Google Scholar 

  24. Lappalainen, P., Aasa, R., Malmström, B. G. & Saraste, M. Soluble CuA-binding domain from the Paracoccus cytochrome c oxidase. J. Biol. Chem. 268, 26416–26421 (1993)

    CAS  PubMed  Google Scholar 

  25. Von Wachenfeldt, C., de Vries, S. & Van der Oost, J. The CuA site of the caa 3-type oxidase of Bacillus subtilis is a mixed-valence binuclear copper center. FEBS Lett. 340, 109–113 (1994)

    CAS  Article  Google Scholar 

  26. Slutter, C. E. et al. Water-soluble, recombinant CuA-domain of the cytochrome ba 3 subunit II from Thermus thermophilus . Biochemistry 35, 3387–3395 (1996)

    CAS  Article  Google Scholar 

  27. Eriste, E., Kruusel, K., Palumaa, P., Jönrvall, H. & Sillard, R. Purification of recombinant human apometallothionein-3 and reconstitution with zinc. Protein Expr. Purif. 31, 161–165 (2003)

    CAS  Article  Google Scholar 

  28. Voronova, A. et al. Cox17, a copper chaperone for cytochrome c oxidase: expression, purification, and formation of mixed disulphide adducts with thiol reagents. Protein Expr. Purif. 53, 138–144 (2007)

    CAS  Article  Google Scholar 

  29. Banci, L. et al. A structural-dynamical characterization of human Cox17. J. Biol. Chem. 283, 7912–7920 (2008)

    CAS  Article  Google Scholar 

  30. Hallewell, R. A. et al. Genetically engineered polymers of human CuZn superoxide dismutase. J. Biol. Chem. 264, 5260–5268 (1989)

    CAS  PubMed  Google Scholar 

  31. Krezel, A. & Maret, W. Thionein/metallothionein control Zn(II) availability and the activity of enzymes. J. Biol. Inorg. Chem. 13, 401–409 (2008)

    CAS  Article  Google Scholar 

  32. Hitomi, Y., Outten, C. E. & O’Halloran, T. V. Extreme zinc-binding thermodynamics of the metal sensor/regulator protein, ZntR. J. Am. Chem. Soc. 123, 8614–8615 (2001)

    CAS  Article  Google Scholar 

  33. Palumaa, P., Kangur, L., Voronova, A. & Sillard, R. Metal-binding mechanism of Cox17, a copper chaperone for cytochrome c oxidase. Biochem. J. 382, 307–314 (2004)

    CAS  Article  Google Scholar 

  34. Presta, A., Green, A. R., Zelazowski, A. & Stillman, M. J. Copper binding to rabbit liver metallothionein. Formation of a continuum of copper(I)-thiolate stoichiometric species. Eur. J. Biochem. 227, 226–240 (1995)

    CAS  Article  Google Scholar 

  35. Saks, V. A., Kupriyanov, V. V., Elizarova, G. V. & Jacobus, W. E. Studies of energy transport in heart cells. The importance of creatine kinase localization for the coupling of mitochondrial phosphorylcreatine production to oxidative phosphorylation. J. Biol. Chem. 255, 755–763 (1980)

    CAS  PubMed  Google Scholar 

  36. Leavesley, H. B., Prabhakaran, K., Borowitz, J. L. & Isom, G. E. Interaction of cyanide and nitric oxide with cytochrome c oxidase: implications for acute cyanide toxicity. Toxicol. Sci. 101, 101–111 (2008)

    CAS  Article  Google Scholar 

  37. Yonetani, T. & Ray, G. S. Kinetics of the aerobic oxidation of ferrocytochrome c by cytochrome c oxidase. J. Biol. Chem. 240, 3392–3398 (1965)

    CAS  PubMed  Google Scholar 

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This work was supported by grants from the Estonian Science Foundation project 7191, the Estonian Ministry of Education and Research (grant SF0140055s08), by the SPINE-II-COMPLEXES contract LSHG-CT-2006-031220, by the FIRB PROTEOMICA MIUR contract RBRN07BMCT and by a World Federation of Scientists scholarship to K.Z. We thank K. Saar for preparative work with rat mitochondrial fractions and for measuring the enzymatic activity of CcO, and C. Massagni for preparing the CCS expression plasmid.

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L.B., I.B., S.C.-B., K.Z., P.P. designed the research; K.Z., T.K. and P.P. performed the research. All authors analysed the data and contributed to the writing of the paper.

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Correspondence to Ivano Bertini or Peep Palumaa.

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The authors declare no competing financial interests.

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Supplementary Information

This file contains Supplementary Figures S1-S18 with legends. Figure S1 is a simple schematic summarizing the main findings in the paper. Figures S2-18 report ESI-MS spectra performed on the copper proteins, the fittings of the ESI-MS data to obtain the apparent dissociation constants and kinetics data on demetallation processes of copper enzymes. (PDF 3420 kb)

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Banci, L., Bertini, I., Ciofi-Baffoni, S. et al. Affinity gradients drive copper to cellular destinations. Nature 465, 645–648 (2010).

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