Denaturation of thermophilic ferricytochrome c-552 by acid, guanidine hydrochloride, and heat

Abstract

The denaturation of Thermus thermophilus cytochrome c-552 by acid, guanidine hydrochloride, and heat was studied by measuring the changes in absorption and circular dichroism. Cytochrome c-552 was remarkably resistant to acid; the pK of the transition from the low- to the high-spin form was roughly 0.3. The effect of guanidine hydrochloride on the heme iron-methionine bond of Thermus and horse cytochromes c was also investigated; a comparison of the free-energy changes for the displacement of the bond indicated that the coordination in cytochrome c-552 is highly stable. The spectra of guanidine hydrochloride unfolded cytochrome c-552 were dependent on the pH; the titration curve showed the presence of a cooperative single transition of pK = 4.7, with a one-proton dissociation, suggesting the ionization of a histidine residue. In the presence of guanidine hydrochloride, the influence of the heat on the ligand bond in cytochrome c-552 was studied. The van't Hoff plots of the reaction were biphasic. The enthalpy changes in the higher temperature range were independent on the guanidine hydrochloride concentration, while those in the lower range were not.