Evidence for an Unusual Electronic Structure of Wheat Germ Peroxidase Compound I

Oxidized states of wheat germ peroxidase isozyme C2 (WGP C2) were investigated by means of electronic absorption spectroscopy. Addition of one molar equivalent of H2O2to ferric WGP C2 led to the formation of an oxidized species with an absorption spectrum very similar to that of peroxidase compound II, with a Soret maximum at 411 nm and visible maxima at 523 and 553 nm. The transformation took place with an isosbestic point at 409 nm. Stopped flow spectroscopy showed no inflection points for the formation of this species when it was registered at 420 nm, and we could verify the persistence of the isosbestic point from 20 ms to 10 s. The oxidized species decays spontaneously to ferric enzyme in a double-exponential manner. By adding excess H2O2to the system we obtained an inactive derivative identical to horseradish peroxidase P-670. In the presence of one equivalent of reducing substrate and excess H2O2compound III was formed. The results so indicate that the species obtained in the reaction of WGP C2 with equimolecular amounts of H2O2is compound I. The resulting compound I spectrum was identical to that of cytochrome c peroxidase, suggesting the formation of a protein radical rather than the typical π cation radical, a feature which had not been described before for a plant peroxidase.