Low spin states of microperoxidases produced by inter- and intra-peptide chain sixth ligands: effect of pH and the oligopeptide type.

The low spin states of microperoxidases (MP)-8, -9 and -9 N-acetylated (N-Ac) were characterized using UV-visible, circular dichroism, and electron paramagnetic resonance spectroscopies over the 6.0-12.0 pH range. The first MP-8 alkaline transition (pK(a)=8.53) produced hemepeptide aggregates in the low spin state in which a water molecule was replaced by the peptide chain N-terminal group of a neighboring MP-8 molecule. Higher pH led to the deprotonation of the MP-8 histidine imidazole ring (pK(a)=10.37) at the fifth coordination position. This MP-8 species was in equilibrium with a high spin state aggregate in which OH(-) replaced histidinate, the histidinate becoming the heme iron sixth ligand in a neighboring MP-8 molecule. In a similar way to the N-AcMP-8, the low spin state of N-AcMP-9 was produced by the deprotonation of the water molecule (pK(a)=9.6) situated at the sixth coordination position of the heme iron. Up to pH 8.5, the low spin states of MP-9 were aggregates in which the alpha-amino group of Lys13 replaced water at the sixth coordination position of a neighboring MP-9 molecule. Above pH 8.5, the epsilon-amino groups of Lys13 established intra-chain coordination and impaired the formation of aggregates. Such intra-chain interaction in MP9 was supported by molecular dynamics simulation. These MP-9 monomers might also exhibit OH(-) or histidinate at the fifth coordination position.