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

Abstract

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.

Keywords

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

Abbreviations

6cLS

Six-coordinated low spin

CV

Cyclic voltammetry

MU

11-Mercapto-1-undecanol

MUA

11-Mercapto-1-undecanoic acid

SAM

Self-assembled monolayer

SCE

Saturated calomel electrode

SERR

Surface-enhanced resonance Raman

ycc

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

Notes

Acknowledgments

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)

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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

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