Thermodynamic Intermediates of the Alkaline III→IV Transition in Ferricytochrome c Probed by 695 nm Charge Transfer Band

Abstract

The 695 nm band in the spectrum of the native state (III) of ferricytochrome c has recently been shown to be composed of different sub-bands which reflect different conformational substates (CS) of the functional pivotal Fe-M80 linkage. In order to explore the influence of the alkaline III→IV transition on this CSs we measured and analyzed the absorption and CD profile of this band as a function of pH between pH 7 and 10 at high (50mM) and low anion concentration for horse heart (hh) cytochrome c. Additional measurements on yeast cytochrome c were performed at low anion concentrations. The titration curves of the two dominating sub-bands are clearly biphasic at low anion concentrations and reflect effective pK-values of 8.5 and 9.65. On the contrary, the titrations curves obtained at high anion concentrations are monophasic; the effective pK being 9.23. The thermodynamic parameters (i.e. pK and the Hill coefficient as a measure of cooperativity) are slightly different for the two CSs. We fitted the data obtained at low anion concentration with a model, which assigns the two effective pK-values to two different alkaline states IV1 and IV2, in which M80 is replaced by K73 and K79, respectively. This model did not reproduce our data well in contrast to an alternative model which assumes only one species with two protonation sites. This suggests that the state populated upon the pK= 8.5 protonation can be interpreted as a thermodynamic intermediate of the III→IV transition. This notion is consistent with our observation, that the 695 nm titration curves of the y-cytochrome c mutants K79R and K73V are still mostly biphasic at low ionic strength.