Determination of reduction potential of an engineered CuA azurin by cyclic voltammetry and spectrochemical titrations

Original Article


The reduction potentials of an engineered CuA azurin in its native and thermally denatured states have been determined using cyclic voltammetry and spectrochemical titrations. Using a 4,4′-dipyridyl disulfide modified gold electrode, the reduction potentials of native and thermally denatured CuA azurin are the same within the experimental error (422±5 and 425±5 mV vs. NHE, respectively, in 50 mM ammonium acetate buffer, pH 5.1, 300 mM NaCl, 25 °C), indicating that the potential is that of a nonnative state. In contrast, using a didodecyldimethylammonium bromide (DDAB) film-pyrolytic graphite edge (PGE) electrode, the reduction potentials of native and thermally denatured CuA azurin have been determined to be 271±7 mV (50 mM ammonium acetate buffer, pH 5.1, 4 °C) and 420±1 mV (50 mM ammonium acetate buffer, pH 5.1, 25 °C), respectively. Spectroscopic redox titration using [Ru(NH3)5Py]2+ resulted in a reduction potential (254±4 mV) (50 mM ammonium acetate buffer, pH 5.1, 4 °C) similar to the value obtained using the DDAB film-PGE electrochemical method. Complete reoxidation of [Ru(NH3)5Py]2+-reduced CuA azurin is also consistent with the conclusion that this spectrochemical titration method using [Ru(NH3)5Py]2+ measures the reduction potential of native CuA azurin.


Copper Electron transfer Cytochrome c oxidase 



cytochrome c oxidase


nitrous oxide reductase


electron transfer


cyclic voltammetry


normal hydrogen electrode


didodecyldimethylammonium bromide


pyrolytic graphite edge



This material is based upon work supported by the National Science Foundation (CHE-0139203). We thank Drs. Kevin R. Hoke and Christophe Léger, and Professors Fraser A. Armstrong, Michael G. Hill and Sean J. Elliott for advice on electrochemistry.


  1. 1.
    Gray HB, Malmström BG, Williams RJP (2000) J Biol Inorg Chem 5:551–559Google Scholar
  2. 2.
    Beinert H (1997) Eur J Biochem 245:521–532Google Scholar
  3. 3.
    Vila AJ, Fernández CO (2001) In: Bertini I, Sigel A, Sigel H (eds) Handbook on metalloproteins. Marcel Dekker, New York, pp 813–856Google Scholar
  4. 4.
    Solomon EI, Randall DW, Glaser T (2000) Coord Chem Rev 200–202:595–632Google Scholar
  5. 5.
    Lu Y (2004) In: Que L Jr, Tolman WB (eds) Biocoordination chemistry. In: McCleverty JA, Meyer TJ (eds) Comprehensive coordination chemistry II: from biology to nanotechnology, vol 8. Elsevier, Oxford, pp 91–122Google Scholar
  6. 6.
    Colman PM, Freeman HC, Guss JM, Murata M, Norris VA, Ramshaw JAM, Venkatappa MP (1978) Nature 272:319–324Google Scholar
  7. 7.
    Adman ET, Stenkamp RE, Sieker LC, Jensen LH (1978) J Mol Biol 123:35–47Google Scholar
  8. 8.
    Nar H, Messerschmidt A, Huber R, van der Kamp M, Canters GW (1991) J Mol Biol 221:765–772Google Scholar
  9. 9.
    Adman ET, Jensen LH (1981) Isr J Chem 21:8–12Google Scholar
  10. 10.
    Lowery MD, Solomon EI (1992) Inorg Chim Acta 198–200:233–243Google Scholar
  11. 11.
    Iwata S, Ostermeier C, Ludwig B, Michel H (1995) Nature 376:660–669Google Scholar
  12. 12.
    Wilmanns M, Lappalainen P, Kelly M, Sauer-Eriksson E, Saraste M (1995) Proc Natl Acad Sci USA 92:11955–11959Google Scholar
  13. 13.
    Robinson H, Ang MC, Gao Y-G, Hay MT, Lu Y, Wang AHJ (1999) Biochemistry 38:5677–5683Google Scholar
  14. 14.
    Williams PA, Blackburn NJ, Sanders D, Bellamy H, Stura EA, Fee JA, McRee DE (1999) Nat Struct Biol 6:509–516Google Scholar
  15. 15.
    Brown K, Tegoni M, Prudencio M, Pereira AS, Besson S, Moura JJ, Moura I, Cambillau C (2000) Nat Struct Biol 7:191–195Google Scholar
  16. 16.
    Steffens GJ, Buse G (1979) Hoppe-Seyler’s Z Physiol Chem 360:613–619Google Scholar
  17. 17.
    van der Oost J, Lappalainen P, Musacchio A, Warne A, Lemieux L, Rumbley J, Gennis RB, Aasa R, Pascher T, Malmström BG, Saraste M (1992) EMBO J 11:3209–3217Google Scholar
  18. 18.
    Dennison C, Vijgenboom E, de Vries S, van der Oost J, Canters GW (1995) FEBS Lett 365:92–94Google Scholar
  19. 19.
    Hay MT, Richards JH, Lu Y (1996) Proc Natl Acad Sci USA 93:461–464Google Scholar
  20. 20.
    Hay MT, Ang MC, Gamelin DR, Solomon EI, Antholine WE, Ralle M, Blackburn NJ, Massey PD, Wang X, Kwon AH, Lu Y (1998) Inorg Chem 37:191–198Google Scholar
  21. 21.
    Gamelin DR, Randall DW, Hay MT, Houser RP, Mulder TC, Canters GW, de Vries S, Tolman WB, Lu Y, Solomon EI (1998) J Am Chem Soc 120:5246–5263Google Scholar
  22. 22.
    Blackburn NJ, Ralle M, Sanders D, Fee JA, de Vries S, Houser RP, Tolman WB, Hay MT, Lu Y (1998) In: Solomon EI, Hodgson KO (eds) Spectroscopic methods in bioinorganic chemistry, no 692. ACS, Washington, DC, pp 241–261Google Scholar
  23. 23.
    Farver O, Lu Y, Ang MC, Pecht I (1999) Proc Natl Acad Sci USA 96:899–902Google Scholar
  24. 24.
    Pascher T, Karlsson BG, Nordling M, Malmström BG, Vänngård T (1993) Eur J Biochem 212:289–296Google Scholar
  25. 25.
    Sun D, Wang X, Davidson VL (2002) Arch Biochem Biophys 404:158–162Google Scholar
  26. 26.
    Immoos C, Hill MG, Sanders D, Fee JA, Slutter CE, Richards JH, Gray HB (1996) J Biol Inorg Chem 1:529–531Google Scholar
  27. 27.
    Lappalainen P, Aasa R, Malmström BG, Saraste M (1993) J Biol Chem 268:26416–26421Google Scholar
  28. 28.
    Jeuken LJC, Armstrong FA (2001) J Phys Chem B 105:5271–5282Google Scholar
  29. 29.
    Bard AJ, Faulkner LR (1980) Electrochemical methods: fundamentals and applications. Wiley, New YorkGoogle Scholar
  30. 30.
    Cummins D, Gray HB (1977) J Am Chem Soc 99:5158–5167Google Scholar
  31. 31.
    Goldberg M, Pecht I (1976) Biochemistry 15:4197–4208Google Scholar
  32. 32.
    Rusling JF, Nassar AEF (1993) J Am Chem Soc 115:11891–11897Google Scholar
  33. 33.
    Wittung-Stafshede P, Hill MG, Gomez E, Di Bilio AJ, Karlsson BG, Leckner J, Winkler JR, Gray HB, Malmström BG (1998) J Biol Inorg Chem 3:367–370Google Scholar
  34. 34.
    Hwang HJ, Lu Y (2004) J Inorg Biochem 98:797–802Google Scholar
  35. 35.
    Ford P, Rudd DFP, Gaunder R, Taube H (1968) J Am Chem Soc 90:1187–1194Google Scholar
  36. 36.
    Lim HS, Barclay DJ, Anson FC (1972) Inorg Chem 11:1460–1466Google Scholar
  37. 37.
    Lide DR, Frederikse HPR (1997) CRC handbook of chemistry and physics, 78th edn. CRC Press, New YorkGoogle Scholar

Copyright information

© SBIC 2004

Authors and Affiliations

  1. 1.Department of ChemistryUniversity of Illinois at Urbana-ChampaignUrbanaUSA

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