JBIC Journal of Biological Inorganic Chemistry

, Volume 13, Issue 6, pp 899–907 | Cite as

Electron paramagnetic resonance characterization of the copper-resistance protein PcoC from Escherichia coli

  • Simon C. DrewEmail author
  • Karrera Y. Djoko
  • Lianyi Zhang
  • Melissa Koay
  • John F. Boas
  • John R. Pilbrow
  • Zhiguang Xiao
  • Kevin J. Barnham
  • Anthony G. Wedd
Original Paper


Continuous-wave and pulsed electron paramagnetic resonance have been applied to the study of the CuII site of the copper-resistance protein PcoC from Escherichia coli and certain variant forms. Electron spin echo envelope modulation (ESEEM) experiments confirm the presence of two histidine ligands, His1 and His92, at the CuII site of wild-type PcoC, consistent with the available X-ray crystallographic data for the homolog CopC (67% sequence identity) from Pseudomonas syringae pv. tomato. The variants H1F and H92F each lack one of the histidine residues close to the CuII site. The ESEEM data suggest that the surviving histidine residue remains as a ligand. The nA variant features an extra alanine residue at the N terminus, which demotes the His1 ligand to position 2. At least one of the two histidine residues is bound at the CuII site in this form. Simulation of the 14N superhyperfine structure in the continuous-wave spectra confirms the presence of at least three nitrogen-based ligands at the CuII sites of the wild-type, H92F and nA forms, while the H1F variant has two nitrogen ligands. The spectra of wild-type form can be fitted adequately with a 3N or a 4N model. The former is consistent with the crystal structure of the CopC homolog, where His1 acts as a bidentate ligand. The latter raises the possibility of an additional unidentified nitrogen ligand. The markedly different spectra of the H1F and nA forms compared with the wild-type and H92F proteins further highlight the integral role of the N-terminal histidine residue in the high-affinity CuII site of PcoC.


Copper transport Copper proteins Electron paramagnetic resonance Electron spin echo envelope modulation Hyperfine sublevel correlation experiment 



Continuous wave


Electron paramagnetic resonance


Electron spin echo envelope modulation


Hyperfine sublevel correlation experiment



We thank Australian Research Council for financial support via grant A29930204. S.C.D. and K.J.B. are funded in part by the National Health and Medical Research Council.

Supplementary material

775_2008_377_MOESM1_ESM.pdf (313 kb)
Supporting Information (PDF 313 kb)


  1. 1.
    Rensing C, Grass G (2003) FEMS Microbiol Rev 27:197–213PubMedCrossRefGoogle Scholar
  2. 2.
    Tetaz TJ, Luke RKJ (1983) J Bacteriol 154:1263–1268PubMedGoogle Scholar
  3. 3.
    Rouch D, Camakaris J, Lee BT, Luke RK (1985) J Gen Microbiol 131:939–943PubMedGoogle Scholar
  4. 4.
    Brown NL, Barrett SR, Camakaris J, Lee BT, Rouch DA (1995) Mol Microbiol 17:1153–1166PubMedCrossRefGoogle Scholar
  5. 5.
    Cooksey DA (1994) FEMS Microbiol Rev 14:381–386PubMedCrossRefGoogle Scholar
  6. 6.
    Lee SM, Grass G, Rensing C, Barrett SR, Yates CJD, Stoyanov JV, Brown NL (2002) Biochem Biophys Res Commun 295:616–620PubMedCrossRefGoogle Scholar
  7. 7.
    Puig S, Thiele DJ (2002) Curr Opin Chem Biol 6:171–180PubMedCrossRefGoogle Scholar
  8. 8.
    Huffman DL, Huyett J, Outten FW, Doan PE, Finney LA, Hoffman BM, O’Halloran TV (2002) Biochemistry 41:10046–10055PubMedCrossRefGoogle Scholar
  9. 9.
    Arnesano F, Banci L, Bertini I, Thompsett AR (2002) Structure 10:1337–1347PubMedCrossRefGoogle Scholar
  10. 10.
    Arnesano F, Banci L, Bertini I, Mangani S, Thompsett AR (2003) Proc Natl Acad Sci USA 100:3814–3819PubMedCrossRefGoogle Scholar
  11. 11.
    Koay M, Zhang L, Yang B, Maher MJ, Xiao Z, Wedd AG (2005) Inorg Chem 44:5203–5205PubMedCrossRefGoogle Scholar
  12. 12.
    Zhang L, Koay M, Maher MJ, Xiao Z, Wedd AG (2006) J Am Chem Soc 128:5834–5850PubMedCrossRefGoogle Scholar
  13. 13.
    Gaggelli E, Kozlowski H, Valensin D, Valensin G (2006) Chem Rev 106:1995–2044PubMedCrossRefGoogle Scholar
  14. 14.
    Sankararamakrishnan R, Verma S, Kumar S (2005) Proteins 58:211–221PubMedCrossRefGoogle Scholar
  15. 15.
    Harford C, Sarkar B (1997) Acc Chem Res 30:123–130CrossRefGoogle Scholar
  16. 16.
    Djoko KY, Xiao Z, Wedd AG (2008) Chembiochem (in press)Google Scholar
  17. 17.
    Wernimont AK, Huffman DL, Finney LA, Demeler B, O’Halloran TV, Rosenzweig AC (2003) J Biol Inorg Chem 8:185–194PubMedCrossRefGoogle Scholar
  18. 18.
    Peariso K, Huffman DL, Penner-Hahn JE, O’Halloran TV (2003) J Am Chem Soc 125:342–343PubMedCrossRefGoogle Scholar
  19. 19.
    Djoko KY, Xiao Z, Huffman DL, Wedd AG (2007) Inorg Chem 46:4560–4568PubMedCrossRefGoogle Scholar
  20. 20.
    Hanson GR, Gates KE, Noble CJ, Griffin M, Mitchell A, Benson S (2004) J Inorg Biochem 98:903–916PubMedCrossRefGoogle Scholar
  21. 21.
    Peisach J, Blumberg WE (1974) Arch Biochem Biophys 165:691–698PubMedCrossRefGoogle Scholar
  22. 22.
    Deligiannakis Y, Louloudi M, Hadjiliadis N (2000) Coord Chem Rev 204:1–112CrossRefGoogle Scholar
  23. 23.
    McCracken J, Pember S, Benkovic SJ, Villafranca JJ, Miller RJ, Peisach J (1988) J Am Chem Soc 110:1069–1074CrossRefGoogle Scholar
  24. 24.
    Schweiger A, Jeschke G (2001) principles of pulse electron paramagnetic resonance. Oxford University Press, OxfordGoogle Scholar
  25. 25.
    Mims WB, Peisach J (1978) J Chem Phys 69:4921–4930CrossRefGoogle Scholar
  26. 26.
    Jeschke G (1996) Ph.D thesis, Swiss Federal Institute of Technology, Sect 6.3.2Google Scholar
  27. 27.
    Van Doorslaer S, Sierra GA, Schweiger A (1999) J Magn Reson 136:152–158PubMedCrossRefGoogle Scholar
  28. 28.
    Jin H, Thomann H, Coyle CL, Zumft WG (1989) J Am Chem Soc 111:4262–4269CrossRefGoogle Scholar
  29. 29.
    Slutter CE, Gromov I, Epel B, Pecht I, Richards JH, Goldfarb D (2001) J Am Chem Soc 123:5325–5336PubMedCrossRefGoogle Scholar

Copyright information

© SBIC 2008

Authors and Affiliations

  • Simon C. Drew
    • 1
    • 3
    Email author
  • Karrera Y. Djoko
    • 2
  • Lianyi Zhang
    • 2
  • Melissa Koay
    • 2
  • John F. Boas
    • 3
  • John R. Pilbrow
    • 3
  • Zhiguang Xiao
    • 2
  • Kevin J. Barnham
    • 1
  • Anthony G. Wedd
    • 2
  1. 1.Department of Pathology, Bio21 Molecular Science and Biotechnology InstituteUniversity of MelbourneParkvilleAustralia
  2. 2.School of Chemistry, Bio21 Molecular Science and Biotechnology InstituteUniversity of MelbourneParkvilleAustralia
  3. 3.School of PhysicsMonash UniversityClaytonAustralia

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