JBIC Journal of Biological Inorganic Chemistry

, Volume 15, Issue 8, pp 1233–1242 | Cite as

The impact of urea-induced unfolding on the redox process of immobilised cytochrome c

  • Stefano Monari
  • Diego Millo
  • Antonio Ranieri
  • Giulia Di Rocco
  • Gert van der Zwan
  • Cees Gooijer
  • Silvia Peressini
  • Claudio Tavagnacco
  • Peter Hildebrandt
  • Marco Borsari
Original Paper


We have studied the effect of urea-induced unfolding on the electron transfer process of yeast iso-1-cytochrome c and its mutant K72AK73AK79A adsorbed on electrodes coated by mixed 11-mercapto-1-undecanoic acid/11-mercapto-1-undecanol self-assembled monolayers. Electrochemical measurements, complemented by surface enhanced resonance Raman studies, indicate two distinct states of the adsorbed proteins that mainly differ with respect to the ligation pattern of the haem. The native state, in which the haem is axially coordinated by Met80 and His18, displays a reduction potential that slightly shifts to negative values with increasing urea concentration. At urea concentrations higher than 6 M, a second state prevails in which the Met80 ligand is replaced by an additional histidine residue. This structural change in the haem pocket is associated with an approximately 0.4 V shift of the reduction potential to negative values. These two states were found for both the wild-type protein and the mutant in which lysine residues 72, 73 and 79 had been substituted by alanines. The analysis of the reduction potentials, the reaction enthalpies and entropies as well as the rate constants indicates that these three lysine residues have an important effect on stabilising the protein structure in the adsorbed state and facilitating the electron transfer dynamics.


Unfolding Cytochrome c Electron transfer process Surface-enhanced resonance Raman Self-assembled monolayer 



Six-coordinated low spin


Cyclic voltammetry




11-Mercapto-1-undecanoic acid


Self-assembled monolayer


Saturated calomel electrode


Surface-enhanced resonance Raman


Recombinant non-trimethylated Saccharomyces cerevisiae iso-1-cytochrome c



We gratefully acknowledge Murat Sezer for supporting the SERR spectroscopy measurements in Berlin. This work was performed with financial support from MIUR (COFIN 2007, protocollo 20079Y9578_002, Bioelettrochimica: trasferimento di carica in sistemi di rilevanza biologica), the University of Modena and Reggio Emilia, the Deutsche Forschungsgemeinschaft (Sfb498), the Alexander von Humboldt Foundation (D.M.) and the European Community Access to Research Infrastructures Action of The Improving Human Potential (contract no. HPRI-CT-1999-00064) (A.R.).

Supplementary material

775_2010_681_MOESM1_ESM.pdf (90 kb)
Supplementary material 1 (PDF 89 kb)


  1. 1.
    Messerschmidt A, Huber R, Poulos T, Wieghardt K (eds) (2001) Handbook of metalloproteins, vol 1. Wiley, ChichesterGoogle Scholar
  2. 2.
    Scott RA, Mauk GA (eds) (1996) Cytochrome c: a multidisciplinary approach. University Science Books, SausalitoGoogle Scholar
  3. 3.
    Moore GR, Pettigrew GW (1990) Cytochromes c: evolutionary, structural, and physicochemical aspects. Springer, BerlinGoogle Scholar
  4. 4.
    Kagan VE, Bayr HA, Belikova NA, Kapralov O, Tyurina YY, Jang J, Stoyanovsky DA, Wipf P, Kochanek PM, Greenberger JS, Pitt B, Shvedova AA, Borisenko G (2009) Free Radic Biol Med 46:1439–1453CrossRefPubMedGoogle Scholar
  5. 5.
    Bond AM (1994) Inorg Chim Acta 226:293–340CrossRefGoogle Scholar
  6. 6.
    Hill HAO, Hunt NI (1993) Methods Enzymol 227:501–522CrossRefPubMedGoogle Scholar
  7. 7.
    Armstrong FA (1990) Struct Bonding 72:137–222CrossRefGoogle Scholar
  8. 8.
    Armstrong FA, Hill HAO, Walton NJ (1986) Q Rev Biophys 18:261–322CrossRefGoogle Scholar
  9. 9.
    Murgida DH, Hildebrandt P (2008) Chem Soc Rev 37:937–945CrossRefPubMedGoogle Scholar
  10. 10.
    Bryngelson JD, Onuchic JN, Socci ND, Wolynes PG (1995) Proteins 21:167–195CrossRefPubMedGoogle Scholar
  11. 11.
    Dobson CM, Sali A, Karplus M (1998) Angew Chem Int Ed 37:868–893CrossRefGoogle Scholar
  12. 12.
    Dobson CM, Karplus M (1999) Curr Opin Struct Biol 9:92–101CrossRefPubMedGoogle Scholar
  13. 13.
    Yeh SR, Rousseau DL (1998) Nat Struct Biol 5:222–228CrossRefPubMedGoogle Scholar
  14. 14.
    Xu Y, Mayne L, Englander SW (1998) Nat Struct Biol 5:774–778CrossRefPubMedGoogle Scholar
  15. 15.
    Russell BS, Melenkivitz R, Bren KL (2000) Proc Natl Acad Sci USA 97:8312–8317CrossRefPubMedGoogle Scholar
  16. 16.
    Myer YP, MacDonald LH, Verma BC, Pande A (1980) Biochemistry 19:199–207CrossRefPubMedGoogle Scholar
  17. 17.
    Yeh SR, Han SW, Rousseau DL (1998) Acc Chem Res 31:727–736CrossRefGoogle Scholar
  18. 18.
    Zhou J, Zheng J, Jiang S (2004) J Phys Chem B 108:17418–17424CrossRefGoogle Scholar
  19. 19.
    Xu J, Bowden EF (2006) J Am Chem Soc 128:6813–6822CrossRefPubMedGoogle Scholar
  20. 20.
    Battistuzzi G, Borsari M, De Rienzo F, Di Rocco G, Ranieri A, Sola M (2007) Biochemistry 46:1694–1702CrossRefPubMedGoogle Scholar
  21. 21.
    Rosell FI, Ferrer JC, Mauk AG (1998) J Am Chem Soc 120:11234–11245CrossRefGoogle Scholar
  22. 22.
    Pollock WBR, Rosell FI, Twitchett MB, Dumont ME, Mauk AG (1998) Biochemistry 37:6124–6131CrossRefPubMedGoogle Scholar
  23. 23.
    Cutler RJ, Pielak GJ, Mauk AG, Smith M (1987) Protein Eng 1:95–99CrossRefPubMedGoogle Scholar
  24. 24.
    Liang N, Mauk AG, Pielak GJ, Johnson JA, Smith M, Hoffmann B (1988) Science 240:311–313CrossRefPubMedGoogle Scholar
  25. 25.
    Battistuzzi G, Borsari M, Sola M, Francia F (1997) Biochemistry 36:16247–16258CrossRefPubMedGoogle Scholar
  26. 26.
    Battistuzzi G, Borsari M, Bortolotti CA, Di Rocco G, Ranieri A, Sola M (2007) J Phys Chem B 111:10281–10287CrossRefPubMedGoogle Scholar
  27. 27.
    Yee EL, Cave RJ, Guyer KL, Tyma PD, Weaver MJ (1979) J Am Chem Soc 101:1131–1137CrossRefGoogle Scholar
  28. 28.
    Yee EL, Weaver MJ (1980) Inorg Chem 19:1077–1079CrossRefGoogle Scholar
  29. 29.
    Song S, Clark RA, Bowden EF, Tarlov MJ (1993) J Phys Chem 97:6564–6572CrossRefGoogle Scholar
  30. 30.
    Weaver MJ (1979) J Phys Chem 13:1748–1757CrossRefGoogle Scholar
  31. 31.
    Bonifacio A, Millo D, Gooijer C, Boegschoten R, van der Zwan G (2004) Anal Chem 76:1529–1531CrossRefPubMedGoogle Scholar
  32. 32.
    Feng JJ, Murgida DH, Utesch T, Mroginski MA, Hildebrandt P, Weidinger I (2008) J Phys Chem B 112:15202–15211CrossRefPubMedGoogle Scholar
  33. 33.
    Millo D, Bonifacio A, Ranieri A, Borsari M, Gooijer C, van der Zwan G (2007) Langmuir 23:4340–4345CrossRefPubMedGoogle Scholar
  34. 34.
    Millo D, Bonifacio A, Ranieri A, Borsari M, Gooijer C, van der Zwan G (2007) Langmuir 23:9898–9904CrossRefPubMedGoogle Scholar
  35. 35.
    Battistuzzi G, Borsari M, Sola M (2001) Eur J Inorg Chem 2989–3004Google Scholar
  36. 36.
    Fedurco M, Augustynski J, Indiani C, Smulevich G, Antalík M, Bánó M, Sedlák E, Galscock MC, Dawson JH (2005) J Am Chem Soc 127:7638–7646CrossRefPubMedGoogle Scholar
  37. 37.
    Oellerich S, Wackerbarth H, Hildebrandt P (2002) J Phys Chem B 106:6566–6580CrossRefGoogle Scholar
  38. 38.
    Wackerbarth H, Hildebrandt P (2003) ChemPhysChem 4:714–724CrossRefPubMedGoogle Scholar
  39. 39.
    Murgida DH, Hildebrandt P (2001) J Phys Chem B 105:1578–1586CrossRefGoogle Scholar
  40. 40.
    Hildebrandt P (1991) J Mol Struct 242:379–395CrossRefGoogle Scholar
  41. 41.
    Fedurco M, Augustynski J, Indiani C, Smulevich G, Antalík M, Bánó M, Sedlák E, Galscock MC, Dawson JH (2004) Biochim Biophys Acta 1703:31–41PubMedGoogle Scholar
  42. 42.
    Bhuyan AK, Udgaonkar JB (2001) J Mol Biol 312:1135–1160CrossRefPubMedGoogle Scholar
  43. 43.
    Pilard R, Haladjian J, Bianco P, Serre P-A, Brabec V (1983) Biophys Chem 17:131–137CrossRefPubMedGoogle Scholar
  44. 44.
    Monari S, Ranieri A, Di Rocco G, van der Zwan G, Peressini S, Tavagnacco C, Millo D, Borsari M (2009) J Appl Electrochem 39:2181–2190CrossRefGoogle Scholar
  45. 45.
    Battistuzzi G, Borsari M, Di Rocco G, Ranieri A, Sola M (2004) J Biol Inorg Chem 9:23–26CrossRefPubMedGoogle Scholar
  46. 46.
    Paggi DA, Martín DF, Kranich A, Hildebrandt P, Martí M, Murgida DH (2009) Electrochim Acta 54:4963–4970CrossRefGoogle Scholar
  47. 47.
    Battistuzzi G, Borsari M, Cowan JA, Ranieri A, Sola M (2002) J Am Chem Soc 124:5315–5324CrossRefPubMedGoogle Scholar
  48. 48.
    Bortolotti CA, Battistuzzi G, Borsari M, Facci P, Ranieri A, Sola M (2006) J Am Chem Soc 128:5444–5451CrossRefPubMedGoogle Scholar
  49. 49.
    Grealis C, Magner E (2003) Langmuir 19:1282–1286CrossRefGoogle Scholar
  50. 50.
    Battistuzzi G, Borsari M, Canters GW, De Waal E, Loschi L, Warmerdam G, Sola M (2001) Biochemistry 40:6707–6712CrossRefPubMedGoogle Scholar
  51. 51.
    Bertrand P, Mbarki O, Asso M, Blanchard L, Guerlesquin F, Tegoni M (1995) Biochemistry 34:11071–11079CrossRefPubMedGoogle Scholar
  52. 52.
    Gunner MR, Alexov E, Torres E, Lipovaca S (1997) J Biol Inorg Chem 2:126–134CrossRefGoogle Scholar
  53. 53.
    Mauk AG, Moore GR (1997) J Biol Inorg Chem 2:119–125CrossRefGoogle Scholar
  54. 54.
    Tezcan FA, Winkler JR, Gray HB (1998) J Am Chem Soc 120:13383–13388CrossRefGoogle Scholar
  55. 55.
    Warshel A, Papazyan A, Muegge I (1997) J Biol Inorg Chem 2:143–152CrossRefGoogle Scholar
  56. 56.
    Banci L, Bertini I, Rosato A, Varani G (1999) J Biol Inorg Chem 4:824–837CrossRefPubMedGoogle Scholar
  57. 57.
    Battistuzzi G, Loschi L, Borsari M, Sola M (1999) J Biol Inorg Chem 4:601–607CrossRefPubMedGoogle Scholar
  58. 58.
    Borsari M, Bellei M, Tavagnacco C, Peressini S, Millo D, Costa G (2003) Inorg Chim Acta 349:182–188CrossRefGoogle Scholar
  59. 59.
    Banci L, Bertini I, Gray HB, Luchinat C, Redding T, Rosato A, Turano P (1997) Biochemistry 36:9867–9877CrossRefPubMedGoogle Scholar
  60. 60.
    Furlan S, La Penna G, Banci L, Mealli C (2007) J Phys Chem B 111:1157–1164CrossRefPubMedGoogle Scholar
  61. 61.
    La Penna G, Furlan S, Banci L (2007) J Biol Inorg Chem 12:180–193CrossRefPubMedGoogle Scholar
  62. 62.
    Mao J, Hauser K, Gunner MR (2003) Biochemistry 42:9829–9840CrossRefPubMedGoogle Scholar
  63. 63.
    Liu L, Guo Q-X (2001) Chem Rev 101:673–695CrossRefPubMedGoogle Scholar
  64. 64.
    Grünwald E, Steel C (1995) J Am Chem Soc 117:5687–5692CrossRefGoogle Scholar
  65. 65.
    Grünwald E (1986) J Am Chem Soc 108:5726–5731CrossRefGoogle Scholar
  66. 66.
    Searle MS, Weatwell MS, Williams DH (1995) J Chem Soc Perkin Trans 2 141–151Google Scholar
  67. 67.
    Rekharsky M, Inoue Y (2000) J Am Chem Soc 122:4418–4435CrossRefGoogle Scholar
  68. 68.
    Liu L, Yang C, Guo Q-X (2000) Biophys Chem 84:239–251CrossRefPubMedGoogle Scholar
  69. 69.
    Strazewski P (2002) J Am Chem Soc 124:3546–3554CrossRefPubMedGoogle Scholar
  70. 70.
    Blokzijl W, Engberts JBNF (1993) Angew Chem Int Ed Engl 32:1545–1579CrossRefGoogle Scholar
  71. 71.
    Lumry R, Rajender S (1970) Biopolymers 9:1125–1227CrossRefPubMedGoogle Scholar
  72. 72.
    Krug RR, Hunter WG, Grieger RA (1976) J Phys Chem 80:2335–2351CrossRefGoogle Scholar
  73. 73.
    Ben-Naim A (1975) Biopolymers 14:1337–1355CrossRefGoogle Scholar
  74. 74.
    Lee B, Graziano G (1996) J Am Chem Soc 118:5163–5168CrossRefGoogle Scholar
  75. 75.
    Laviron E (1979) J Electroanal Chem 101:19–28CrossRefGoogle Scholar
  76. 76.
    Millo D, Ranieri A, Gross P, Ly HK, Borsari M, Hildebrandt P, Wuite GJL, Gooijer C, van der Zwan G (2009) J Phys Chem C 113:2861–2866CrossRefGoogle Scholar
  77. 77.
    Murgida DH, Hildebrandt P (2001) J Am Chem Soc 123:4062–4068CrossRefPubMedGoogle Scholar

Copyright information

© SBIC 2010

Authors and Affiliations

  • Stefano Monari
    • 1
  • Diego Millo
    • 2
    • 3
  • Antonio Ranieri
    • 1
  • Giulia Di Rocco
    • 1
  • Gert van der Zwan
    • 3
  • Cees Gooijer
    • 3
  • Silvia Peressini
    • 1
  • Claudio Tavagnacco
    • 4
  • Peter Hildebrandt
    • 2
  • Marco Borsari
    • 1
  1. 1.Department of ChemistryUniversity of Modena and Reggio EmiliaModenaItaly
  2. 2.Max-Volmer-Laboratorium, Sekr. PC14, Institut für ChemieTechnische Universität BerlinBerlinGermany
  3. 3.Laser Centre—Analytical Chemistry and Applied SpectroscopyVrije Universiteit AmsterdamAmsterdamThe Netherlands
  4. 4.Department of ChemistryUniversity of TriesteTriesteItaly

Personalised recommendations