title: The impact of urea-induced unfolding on the redox process of immobilised cytochrome c.
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.
authors.affiliation: Department of Chemistry, University of Modena and Reggio Emilia, Via Campi 183, 41125, Modena, Italy.
authors.forename: Stefano
authors.initials: S
authors.lastname: Monari
authors.forename: Diego
authors.initials: D
authors.lastname: Millo
authors.forename: Antonio
authors.initials: A
authors.lastname: Ranieri
authors.forename: Giulia
authors.initials: G
authors.lastname: Di Rocco
authors.forename: Gert
authors.initials: G
authors.lastname: van der Zwan
authors.forename: Cees
authors.initials: C
authors.lastname: Gooijer
authors.forename: Silvia
authors.initials: S
authors.lastname: Peressini
authors.forename: Claudio
authors.initials: C
authors.lastname: Tavagnacco
authors.forename: Peter
authors.initials: P
authors.lastname: Hildebrandt
authors.forename: Marco
authors.initials: M
authors.lastname: Borsari
mesh_terms: D000327:Adsorption; D045304:Cytochromes c / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004563:Electrochemistry; D004566:Electrodes; D004579:Electron Transport; D004800:Enzymes, Immobilized / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D008958:Models, Molecular; D010084:Oxidation-Reduction; D058767:Protein Unfolding / Q000187:drug effects*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D013059:Spectrum Analysis, Raman; D013499:Surface Properties; D013816:Thermodynamics; D014508:Urea / Q000494:pharmacology*
chemical_list: C014059:CYC1 protein, S cerevisiae; D004800:Enzymes, Immobilized; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; D014508:Urea; D045304:Cytochromes c