The pH dependence of the 695 nm charge transfer band reveals the population of an intermediate state of the alkaline transition of ferricytochrome c at low ion concentrations.

We have measured and analyzed the pH dependence of the 695 nm charge transfer band of horse heart ferricytochrome c as a function of pH between 7.0 and 10.5 at high (50 mM) and low (0.5 mM) phosphate ion concentrations. Our data clearly reveal that the transition from the native state (III) to the two alkaline states (IV) involves two deprotonation steps which cannot be assigned to the two different lysine ligands associated with the two alkaline states. While the respective pK values are rather similar at high phosphate concentrations (9.23 and 9.14), they are clearly different at low anion concentrations (9.65 and 8.5). Apparently, the deprotonation that can be assigned to a pK of 8.5 populates an intermediate state termed III*, in which M80 is still an axial ligand. A comparison of Soret band CD spectra suggests that III* bears some similarity with the recently characterized thermally excited state IIIh. Our data suggest that the current picture of the alkaline transition is incomplete. The obtained results might be of relevance for characterizing the structure of ferricytochrome c bound to anionic phospholipids.