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 O-2 (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 O-2 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 maybe released from the active site and, being more reactive, oxidizes H2O2 to O-2 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. While EDTA binds (K-D = 15 mM) at or near the iodide binding site (Bhattachsaryya et al. (1993) Biochem. J. 289, 575-580), I+ binds to HRP with a K-D value of 20 +/- 7 mu M. I+ binding in the HRP-CN complex(K-D = 20 +/- 8 mu M) indicates that its site is away from the heme iron center. If binding remains unaltered by guaiacol or vice versa, suggesting that I+ binds away from the aromatic donor binding site. As If reduction occurs at a saturating concentration of EDTA, I+ binding at the EDTA site could be excluded. A plot of log KD of I+ binding against various pHs shows the involvement of an ionizable group on the enzyme having pK(a) = 4.8, contributed by an acidic group, deprotonation of which favors I+ binding. Systematic variation of the concentrations of H2O2, EDTA, and I+ under steady state condition yields sets of kinetic parameters containing both kinetic and mechanistic information. Four distinct enzyme species are involved in I+ reduction: native enzyme, compound I, compound I-I+, and compound II-I . complex; and rate constants- for individual steps are calculated. The kinetic experiments support the View that an active enzyme-I+-EDTA ternary complex is formed during I+ reduction by EDTA at the enzyme active site.