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 for iodine reductase activity.
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 (aN = 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.(ABSTRACT TRUNCATED AT 250 WORDS)