Circular dichroism of horseradish peroxidase and its enzyme-substrate compounds

Abstract

The conformations of peroxidase (HNO 2:H 2O 2 oxidoreductase, EC 1.11.1.7), peroxidase-substrate compound I, and peroxidase-substrate compound II were investigated using circular dichroism. Peroxidase has positive circular dichroic bands at 190 and 406 mμ and negative bands at 207, 222, 282, 335, and 370 mμ. The far ultraviolet, circular dichroic spectrum of peroxidase resembles that of α-helical polypeptides. The intensities of the 207- and 222-mμ bands are about 40% of the literature values for completely α-helical polypeptides. A mixture of the two-enzyme-substrate compounds has the same 207- and 222-mμ circular dichroic bands as does free peroxidase, suggesting that all three forms have the same secondary structure. In the Soret region, the formation of enzyme-substrate compounds shifts the wavelengths of the circular dichroic bands; these changes are similar to those known to occur in the absorption bands. Also in the 280-mμ region, the circular dichroism of the enzyme-substrate compounds differs from that of peroxidase, indicating either that the heme moiety contributes to the ultraviolet circular dichroism or that the orientation of an aromatic amino acid residue changes during peroxidatic activity.