Temperature- and pH-dependent changes in the coordination sphere of the heme c group in the model peroxidase N alpha-acetyl microperoxidase-8.

The pH- and temperature-dependent changes in the coordination sphere of the heme c group of N alpha-acetyl microperoxidase-8 (Ac-MP-8) have been studied by examining its optical, resonance Raman, electron paramagnetic resonance, and magnetic circular dichroism spectra. An optical titration indicates that Ac-MP-8 exists in three major ionization forms over the pH 1-12 range that are linked by pK alpha values of approximately 3 and 9. The acid form that is present at pH 1.5 exists as a mixture of five- and six-coordinate high-spin species and most likely has water or buffer ions as axial ligand(s). On titration to pH 7, the His18 residue is deprotonated and becomes the proximal ligand to the iron to give a six-coordinate neutral form that has water as the sixth ligand. This form exists in a thermal high-spin intermediate-spin state equilibrium. On raising the pH to 10, an alkaline form is generated which is predominantly a five-coordinate high-spin species. It is formed by ionization of the proximal His18 residue to its imidazolate form with concomitant dissociation of the water ligand at the sixth site. At concentrations of Ac-MP-8 greater than 10 microM, some six-coordinate low-spin species are formed that are attributed to a dimer in which a His18 residue from a second molecule of Ac-MP-8 coordinates to the sixth site of another to give a bis-His complex. Raising the pH to 11.5 does not produce an appreciable amount of the six-coordinate complex with hydroxide as the sixth ligand. These studies show that Ac-MP-8 is a good water-soluble model for the peroxidases that exhibits minimal aggregation at concentrations below 10 microM in the neutral and alkaline pH regions.