Redox thermodynamics of cytochromes c subjected to urea induced unfolding

Abstract

The thermodynamics of the electron transfer (ET) process for beef heart and yeast cytochromes c and the Lys72Ala/Lys73Ala/Lys79Ala mutant of the latter species subjected to progressive urea-induced unfolding was determined electrochemically. The results indicate the presence of at least three protein forms which were assigned to a low-temperature and a high-temperature HisMet intermediate species and a bis-histidinate form (although the presence of a His-Lys form cannot be excluded). The much lower E°0 value of the bis-histidinate conformer as compared to His-Met ligated species is largely determined by the enthalpic contribution induced by axial ligand substitution. The biphasic E°0 versus T profile for the His-Met species is due to a difference in reduction entropy between the conformers at low and high temperatures. Enthalpy-entropy compensation phenomena for the reduction reaction at varying urea concentration for all the forms of the investigated cytochromes c were addressed and discussed.

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

“…2). Conversely, the respective transitions in solution are found at 3.5 and 3.2 M for ycc and K72AK73AK79A, respectively [44]. Thus, we conclude that Lys72, Lys73 and Lys79 stabilise the protein structure in the adsorbed state.…”

“…The solution contained 5 mM sodium perchlorate and 5 mM phosphate buffer (pH 7, T = 278 K). The spectra were obtained with 413-nm excitation which exhibits the same haem pocket structure as B1 [38], and for cytochrome c in urea-containing solution [36,41,43,44] and is consistent with a change of the ligand from S-Met to N-His [3,35,36,47]. The difference in the reduction potential, DE°0, between these two states is 0.031 V more positive for ycc than for K72AK73AK79A ( Table 2).…”

“…The difference in the reduction potential, DE°0, between these two states is 0.031 V more positive for ycc than for K72AK73AK79A ( Table 2). The DE°0 values for ycc and K72AK73AK79A in solution, however, differ by only 0.014 V [44].…”

“…In contrast, the slight decrease in the E°0 value of wave I upon increasing the urea concentration from 0 to 5 M points to moderate conformational perturbations beyond the level of a ligand exchange and a major rearrangement of the tertiary structure [43,44]. Previous interpretation stressing a Met80 ?…”

“…Lys substitution [15] can be ruled out since K72AK73AK79A lacking all potential Lys ligands reveals essentially the same electrochemical behaviour as ycc. Instead, the increasing negative shift of the reduction potential, which is the result of large and opposing contributions of DS (Table 1) [45], may be due to increasing exposure of the redox centre to the solvent [43,44].…”