The role of hydroxyl radicals in irreversible inactivation of lactoperoxidase by excess H 2O 2: A Spin-trapping/ESR and absorption spectroscopy study

Abstract

H 2O 2 is catalytically metabolized by ferric lactoperoxidase (LPO) → compound (cpd) I → cpd II → ferric LPO cycles. An excess of the substrate, however, is degraded by a ferric LPO → cpd I → cpd II → cpd III → ferrous LPO → ferric LPO cycle. This latter pathway leads to the partial or total irreversible inactivation of the enzyme depending on the excess of H 2O 2 ( H. Jenzer, W. Jones, and H. Kohler (1986) J. Biol. Chem. 261, 15550–15556 ). Spin-trapping/ESR data indicate that in the course of the reaction superoxide ( HO 2 O − 2 ) and hydroxyl radicals (OH.) are formed. Since many substances known to scavenge radicals, such as a spin trap (e.g., 5,5-dimethyl-l-pyrroline- N-oxide) desferrioxamine, albumin, or mannitol, do not prevent enzyme inactivation, we conclude that OH. generation is a site-specific reaction at or near the active center of LPO where bulky scavenger molecules may not be able to penetrate. We suggest the formation of OH. by a Fenton-like reaction between H 2O 2 and the intermediate ferrous state of the enzyme, which substitutes for Fe 2+ in the Fenton reaction. OH. is a powerful oxidant which in turn may attack rapidly the nearest partner available, either H 2O 2 to produce HO. 2 and H 2O, or the prosthetic group to give rise to oxidative cleavage of the porphyrin ring structure of the heme moiety of LPO and thus to the liberation of iron.