Concurrent reduction of iodine and oxidation of EDTA at the active site of horseradish peroxidase: probing the iodine binding site by optical difference spectroscopy and steady state kinetic analysis for the formation of active enzyme-I+-EDTA ternary complex and for iodine reductase activity

Abstract

Horseradish peroxidase (HRP) catalyzes the reduction of iodine to iodide by EDTA with pseudocatalatic degradation of H₂O₂ to O₂ (Banerjee et al., (1986) J. Biol. Chem. 261, 10592-10597; and Banerjee (1989) J. Biol. Chem. 264, 9188-9194). The reduction of iodine (I⁺) is dependent on EDTA concentration and is blocked by spin trap, DMPO, indicating the involvement of free radical species in the reduction process. Incubation of EDTA with both HRP and H₂O₂ results in the appearance of triplet ESR signal of spin-trapped EDTA radical (a^N = 15 G), indicating its one-electron oxidation to a nitrogen-centered monocation radical (N-N⁺). The latter oxidizes H₂O₂ to evolve O₂ and regenerate EDTA. In the presence of I⁺, a ternary complex of compound I-I⁺-EDTA is formed, which generates compound II-I^* complex and both nitrogen-centered dication radical (N⁺-N⁺) through intermolecular electron transfer from EDTA nitrogens. Compound II-I^* complex is further reduced similarly by another molecule of EDTA to form ferric enzyme, I^-, and (N⁺-N⁺).(N⁺-N⁺) the oxidation product of EDTA, which may be released from the active site and, being more reactive, oxidizes H₂O₂ to O₂ at a faster rate to regenerate EDTA. The existence of (N⁺-N⁺) is suggested from the similarity of its ESR signal with that of single nitrogen-centered monocation radical (N-N⁺). EDTA degradation by oxidative decarboxylation due to two-electron oxidation from the same or both nitrogen, atoms is not evident, and EDTA concentration remains the same throughout the reactions.