Conformational stability and dynamics of cytochrome c affect its alkaline isomerization

Abstract

The alkaline isomerization of horse heart ferricytochrome c (cyt c) has been studied by electronic absorption spectroscopy in the presence of the Hofmeister series of anions: chloride, bromide, rhodanide and perchlorate. The anions significantly affect the apparent pK (a) value of the transition in a concentration-dependent manner according to their position in the Hofmeister series. The Soret region of the absorption spectra is not affected by the presence of the salts and shows no significant structural perturbation of the heme crevice. In the presence of perchlorate and rhodanide anions, the cyanide exchange rate between the bulk solvent and the binding site is increased. These results imply higher flexibility of the protein structure in the presence of chaotropic salts. The thermal and isothermal denaturations monitored by differential scanning calorimetry and circular dichroism, respectively, showed a decrease in the conformational stability of cyt c in the presence of the chaotropic salts. A positive correlation between the stability, DeltaG, of cyt c and the apparent pK (a) values that characterize the alkaline transition indicates the presence of a thermodynamic linkage between these conformational transitions. In addition, the rate constant of the cyanide binding and the partial molar entropies of anions negatively correlate with the pK (a) values. This indicates the important role of anion-induced solvent reorganization on the structural flexibility of cyt c in the alkaline transitions.

Rate of oxidative modification of cytochrome c by hydrogen peroxide is modulated by Hofmeister anions

“…However, analysis of the location of these mutations indicates that they play role in both local and global stabilities of cyt c. In fact, an increased local stability (reflected by shifts of acidic and alkaline transitions to more extreme pH values), global stability (increase in temperature of thermal transition), and lowered affinity of cyanide for heme iron strongly indicate a decreased flexibility of protein matrix of the mutated cyt c (Luntz et al 1989;Hickey et al 1991;Schejter et al 1992Schejter et al , 1994. This is in accordance with our recent findings that the pK a of alkaline isomerization of cyt c closely correlates with its local and global stabilities (Tomášková et al 2007). Further analysis indicates that this set of mutations partially or completely perturbs interaction of the polypeptide chain with an internal structural water molecule (Wat166) (Berghuis et al 1994a).…”

Effect of confinement of horse heart cytochrome c and formate dehydrogenase from Candida boidinii on mesoporous carbons on their catalytic activity

“…Indeed, pH is known to have a strong effect in the conformational changes in the heme group of Cyt c, with consequences on the electron transfer kinetic and therefore on the pseudo-peroxidise activity of the protein [ 40 , 44 – 46 ]. Cyt c may exist in solution in five reversible pH-dependent conformational states: I, II, III, IV, and V with pKa values of 0.42, 2.50, 9.35, and 12.76, respectively [ 40 , 47 ].…”

Hofmeister effects: an explanation for the impact of ionic liquids on biocatalysis

“…Indeed, a higher flexibility of cytochrome c in the presence of chaotropic anions has been confirmed by denaturation study of the protein, as monitored by differential scanning calorimetry and circular dichroism (Tomáčková et al, 2007). A plausible explanation for the effect of anions on the activity of Pseudomonas cepacia lipase involves opposite roles played by both kosmotropic and chaotropic anions on the transition state and active site of the enzyme (Bilaničov␣ et al, 2008).…”