Heme-environment of horseradish peroxidase as observed by oxygen-17 superhyperfine interaction in EPR.

The presence of a water ligand on heme-iron in ferric hemoproteins can, in suitable cases, be detected by observing ¹⁷O superhyperfine interaction in the EPR spectra of solutions in H₂¹⁷O. Although no significant superhyperfine interaction is detectable in the EPR spectra of horseradish peroxidase itself, benzo-hydroxamic acid, which forms an outersphere complex with the enzyme analogous to an enzyme-peracid transition state, stabilizes an innersphere water ligand on the heme, as indicated by a ～1.3 gauss Fe³⁺-¹⁷O superhyperfine interaction in the EPR signal at g = 2, in the presence of 34–39% H₂¹⁷O at 8°K. These results indicate that the predominantly pentacoordinate Fe³⁺ ion in horseradish peroxidase is accessible to the solvent and that it acquires a water or hydroxyl ligand in the presence of benzohydroxamic acid.