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

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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.

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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

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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.).

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Authors and Affiliations

  1. Department of Chemistry, University of Modena and Reggio Emilia, Via Campi 183, 41125, Modena, Italy

    Stefano Monari, Antonio Ranieri, Giulia Di Rocco, Silvia Peressini & Marco Borsari

  2. Max-Volmer-Laboratorium, Sekr. PC14, Institut für Chemie, Technische Universität Berlin, Straße des 17. Juni 135, 10623, Berlin, Germany

    Diego Millo & Peter Hildebrandt

  3. Laser Centre—Analytical Chemistry and Applied Spectroscopy, Vrije Universiteit Amsterdam, De Boelelaan 1081, 1081 HV, Amsterdam, The Netherlands

    Diego Millo, Gert van der Zwan & Cees Gooijer

  4. Department of Chemistry, University of Trieste, Via Giorgieri 1, 34127, Trieste, Italy

    Claudio Tavagnacco

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  1. Stefano Monari
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Correspondence to Peter Hildebrandt or Marco Borsari.

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Monari, S., Millo, D., Ranieri, A. et al. The impact of urea-induced unfolding on the redox process of immobilised cytochrome c . J Biol Inorg Chem 15, 1233–1242 (2010). https://doi.org/10.1007/s00775-010-0681-7

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