Interaction of peroxidases with aromatic peracids and alkyl peroxides. Product analysis.

Two limiting structures may be assigned to the primary derivative of peroxidases (Compound I) demanding either (a) total or (b) partial retention of peroxide components. It is now shown that (a) interaction of horseradish peroxidase with m-nitroperbenzoic acid, in 15 mM phosphate, pH 6.2, 25", leads to the stoichiometric formation of the enzymeperoxide derivative, m-nitrobenzoate and 1 proton; and (b) in 5 mM phosphate, pH 6.6, lo’, the conversion of horseradish peroxidase into Compound I with ethyl hydrogen peroxide, is attended by the release of ethanol, but without concomitant prototropic changes. The results establish that horseradish peroxidase (H-peroxidase) Compound I is not an enzyme peroxide complex, but a derivative in which the active site is oxidized. The optical spectra of H-peroxidase and Compound I in 5 mM phosphate, pH 6.6,25”, were re-examined and extended to include previously undefined features in the near infrared absorption region. The spectrum of Compound I is devoid of bands typical of isoporphyrins, suggesting that conversion of the enzyme into Compound I does not involve covalent modification of the prosthetic group.