A systematic analysis of the low-spin to high-spin transition in the ultraviolet and visible absorption spectrum of camphor-bound cytochrome P-450 is presented. The equilibrium depends on the ionization of two groups, the deprotonation of the first one (pK1 = 5.4 at -17 degrees C) leading to an increased content of high-spin type of spectrum, that of the second one (pK2 > 6) to more low-spin type of spectrum. Both protonation processes and their thermodynamic terms are studied under various conditions of salt, solvent and temperature, including subzero temperatures. The basic mechanism of the transition is the same in aqueous and mixed organic solvent, i.e. the protonation of the first group triggers a conformational change of the whole protein. These phenomena are discussed with reference to functional conformational changes during the catalytic cycle of cytochrome P-450.