ELECTRON-PARAMAGNETIC RESONANCE ANALYSES OF HORSERADISH-PEROXIDASE INSITU AND AFTER PURIFICATION

We present EPR spectra of freshly harvested horseradish root, partially purified root extract, and pure horseradish peroxidase isoenzymes A2 and C2 at various values of pH, temperature, and power. They exhibit signals in the g ⋍ 6 and g 5 region, typical of high-spin and quantum mixed-spin heme iron, respectively, with a greater proportion of g 5 signal present in the root and root extract samples than is seen in the spectra of most samples of pure A2 and C2 isoenzymes. The addition of hydroquinone to A2 or C2 causes an increase in the quantum mixed-spin signal and an accompanying decrease in the high-spin signal. Tests for adhering donor in the purified isoenzymes were negative, implying that the quantum mixed-spin signals, g ⋍ 5, originate from free horseradish peroxidase. The addition of hydrogen peroxide decreases the g ⋍ 5 and g ⋍ 6 signals in parallel with each other and the increase in the free radical signal at g ⋍ 2. pH titration of A2 or C2 results in reversible transitions between various high-spin, quantum mixed-spin, and low-spin EPR spectral species. The variation in the relative amounts of quantum mixed-spin and high-spin species monitored in different horseradish peroxidase preparations and the response to conditions of donor and pH show that the protein conformation is sensitive to perturbation imposed upon it during and after purification. The implications of quantum mixed-spin properties for the peroxidase function of the enzyme are discussed in the context of the model for the iron-ligand configuration inferred from magnetic studies of quantum mixed-spin heme proteins. © 1979, American Chemical Society. All rights reserved.