Near-exact enthalpy-entropy compensation governs the thermal unfolding of protonation states of oxidized cytochrome c.

This paper reports the first quantitative analysis of the thermal transitions of all protonation states of oxidized horse heart cytochrome c at low anion concentration. Changes of secondary and tertiary structure were probed by ultraviolet (UV) as well as visible circular dichroism and absorption spectroscopy, respectively. The temperature dependence of spectra were recorded at pH values assignable to a set of different protonation states which encompass the canonical Theorell-Åkesson states and the recently discovered III* state. Our experimental data suggest a two-step process of thermal unfolding for all protonation states. The respective thermodynamic parameters were obtained from a global analysis of the temperature dependence of corresponding visible circular dichroism (CD) and absorption spectra. The results of this analysis revealed a statistically significant enthalpy-entropy compensation with different apparent compensation temperatures for the two consecutive thermal transitions (319 and 357 K). This reflects the narrow distribution of the respective folding temperatures. UVCD spectra suggest that even the thermal transitions of protonation states occupied at acidic and alkaline pH cause only a very modest unfolding of the protein's helical structure. Our data indicate the protonation-induced unfolding at room temperatures predominantly affects the Ω-loops of the protein. The two thermal transitions involve changes of two foldons, i.e. the unfolding of two short β-strand segments (associated with the yellow foldon) followed by the unfolding of the 60' helix (green foldon) that connects the two Ω-loops of the protein. Apparently, intra-backbone hydrogen bonding is strong enough to mostly protect the terminal N- and C-helices from unfolding even at rather extreme conditions.