Formation of two types of low-spin heme in horseradish peroxidase isoenzyme A2 at low temperature

Electronic absorption, resonance Raman and EPR spectra are reported for fer ric horseradish peroxidase isoenzyme A2 at neutral and alkaline pH together with its imidazole complex at 12 K. The data are compared with those obtai ned at room temperature. At neutral pH, lowering the temperature induces co nformational changes with the formation of two types of low-spin hemes, a b is-histidyl type and a hydroxo type. The transition induced by lowering the temperature is accompanied by a change in the orientation of a vinyl subst ituent which appears less conjugated to the porphyrin macrocycle than at ro om temperature. At low temperature the low-spin hemes coexist with a quantu m admired spin species. All the forms are characterized by extremely high r esonance Raman frequencies, indicating a contraction of the core size from that of the room temperature species. At alkaline pH, only one low-spin spe cies is observed at both room and low temperatures, with a hydroxo ligand b ound to the heme iron. The II(Fe-OH) stretching mode has been assigned at 5 12 cm(-1), on the basis of the isotopic shift observed in D2O and (H2O)-O-1 8. This relatively low frequency, together with the anomalous shift observe d in deuterium. indicates that the hydrogen bonds between the oxygen atom a nd the distal residues are stronger than in metmyoglobin, but weaker than t hose of horseradish peroxidase isoenzyme C. This is in agreement with the l ower tetragonality, determined from the EPR g values, of alkaline horseradi sh peroxidase isoenzyme A2 than of metmyoglobin.