The impact of urea-induced unfolding on the redox process of immobilised cytochrome c
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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.
KeywordsUnfolding Cytochrome c Electron transfer process Surface-enhanced resonance Raman Self-assembled monolayer
Six-coordinated low spin
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.).
- 1.Messerschmidt A, Huber R, Poulos T, Wieghardt K (eds) (2001) Handbook of metalloproteins, vol 1. Wiley, ChichesterGoogle Scholar
- 2.Scott RA, Mauk GA (eds) (1996) Cytochrome c: a multidisciplinary approach. University Science Books, SausalitoGoogle Scholar
- 3.Moore GR, Pettigrew GW (1990) Cytochromes c: evolutionary, structural, and physicochemical aspects. Springer, BerlinGoogle Scholar
- 35.Battistuzzi G, Borsari M, Sola M (2001) Eur J Inorg Chem 2989–3004Google Scholar
- 66.Searle MS, Weatwell MS, Williams DH (1995) J Chem Soc Perkin Trans 2 141–151Google Scholar