Conservation of the free energy change of pH-dependent isomerizations in cytochromes c and blue copper proteins

The thermodynamic parameters of the conformational transitions occurring at low pH in blue copper proteins (acid transition), and at high pH in cytochromes c (alkaline transition) have been determined through direct electrochemistry experiments carried out at variable pH and temperature. The former transition involves protonation and detachment from the Cu(I) ion of one histidine ligand (1), whereas the latter leads to a conformer in which the axial methione ligand of the ferriheme is substituted by a surface lysine, the transition being triggered by an as yet unidentified deprotonating residue (2). The blue copper proteins investigated were plastocyanins, R. vernicifera stellacyanin, CBP and T. versutus amicyanin. For all species but CBP the overall conformational change turns out to be exothermic. The entropy change is remarkably species-dependent. It is apparent that the thermodynamic “driving force” for this transition is enthalpic for the plastocyanins and entropic for the phytocyanins. Amicyanin is an intermediate case in which both enthalpic and entropic terms favor the transition. Under the assumption that the transition entropy originates from solvent reorganization effects, which are known to involve compensative enthalpy and entropy changes, the G of the transition would also correspond to the enthalpy change due to bond breaking/formation in the first coordination sphere of the metal and in its immediate environment. Indeed, this term turns out to be very similar for the proteins investigated, in line with the conservation of the Cu(I)-His bond strengths in these species, but amicyanin, for which the greater exotermicity of the transition can be ascribed to peculiar features of the active site. For cytochromes c, we have found that both transition enthalpy and entropy are remarkably species-dependent, following the order: R.pal cytc2 >> beef (horse) heart cytc > yeast iso-1 cytc. Notably, changes in transition enthalpy and entropy among these cytochromes c are compensative and result in small variations in the free energy change of the process (which amounts approximately to +50 kJ mol-1), and consequently in the apparent pKa value. Therefore, enthalpy/entropy compensation phenomena compensation are common to both transitions, and indicate that solvent reorganization effects play an important role in the thermodynamics of the pH-induced conformational changes.