Anion-Specific Effects on the Alkaline State of Cytochrome c

Specific effects of anions on the structure, thermal stability, and peroxidase activity of native (state III) and alkaline (state IV) cytochrome c (cyt c ) have been studied by the UV-VIS absorbance spectroscopy, intrinsic tryptophan fluorescence, and circular dichroism. Thermal and isothermal denaturation monitored by the tryptophan fluorescence and circular dichroism, respectively, implied lower stability of cyt c state IV in comparison with the state III. The p K a value of alkaline isomerization of cyt c depended on the present salts, i.e., kosmotropic anions increased and chaotropic anions decreased p K a (Hofmeister effect on protein stability). The peroxidase activity of cyt c in the state III, measured by oxidation of guaiacol, showed clear dependence on the salt position in the Hofmeister series, while cyt c in the alkaline state lacked the peroxidase activity regardless of the type of anions present in the solution. The alkaline isomerization of cyt c in the presence of 8 M urea, measured by Trp59 fluorescence, implied an existence of a high-affinity non-native ligand for the heme iron even in a partially denatured protein conformation. The conformation of the cyt c alkaline state in 8 M urea was considerably modulated by the specific effect of anions. Based on the Trp59 fluorescence quenching upon titration to alkaline pH in 8 M urea and molecular dynamics simulation, we hypothesize that the Lys79 conformer is most likely the predominant alkaline conformer of cyt c . The high affinity of the sixth ligand for the heme iron is likely a reason of the lack of peroxidase activity of cyt c in the alkaline state.