Proton‐linked effects on the axial coordination in ferric and ferrous horse heart cytochrome <i>c</i>

The effect of pH (between pH 7.0 and 0.0) on the spectroscopic properties of ferric and ferrous derivatives of horse heart cytochrome c (hhcytc-Fe(III) and hhcytc-Fe(II), respectively) in 1.0M phosphate buffer is reported. This study further deepens the investigation of spectroscopic properties of a heme-protein to very low pH (i.e., pH ≈ 0) showing relevant pH-dependent variations of the heme surroundings, involving the heme-Fe atom coordination. For both hhcytc-Fe(III) and hhcytc-Fe(II) a severe weakening of the Fe-Met80 has been detected with a similar pK_a, ranging between 2.5 and 3.0, suggesting a very small effect on the free energy for the protonation of this bond from the oxidation state of the heme's iron. Therefore, in the presence of either CO or NO, it has been possible to detect the formation at acid pH of hhcytc-Fe(II)-CO and hhcytc-Fe(II)-NO forms. On the other hand, the free energy for the protonation of the His18-Fe proximal bond seems dependent on the oxidation state of the heme's iron, since a pK_a ≈ 0.2 is observed for its cleavage in the hhcytc-Fe(III) species, while this bond appears still intact in hhcytc-Fe(II) even at pH ≈ 0. Since during patho-physiological conditions cytochrome c may experience dramatic pH changes of the surroundings, this information turns out of the utmost relevance to explore mechanistic insights of pathological processes, such as redox unbalance and mitochondrial dysfunction, linked to cytochrome c alterations (e.g., neurodegeneration), for which effective modulation of the cytochrome c properties may be of therapeutic relevance.