The role of hydroxyl radicals in irreversible inactivation of lactoperoxidase by excess H2O2. A spin-trapping/ESR and absorption spectroscopy study.

H2O2 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 H2O2 (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 (HO2./O2-) and hydroxyl radicals (OH.) are formed. Since many substances known to scavenge radicals, such as a spin trap (e.g., 5,5-dimethyl-1-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 H2O2 and the intermediate ferrous state of the enzyme, which substitutes for Fe2+ in the Fenton reaction. OH. is a powerful oxidant which in turn may attack rapidly the nearest partner available, either H2O2 to produce HO2. and H2O, 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.