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 iodi de by EDTA with pseudocatalatic degradation of H2O2 to O-2 (Banerjee e t 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 t he involvement of free radical species in the reduction process, Incub ation of EDTA with both HRP and H2O2 results in the appearance of trip let 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 di cation radical (N+-N+) through intermolecular electron transfer from E DTA 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 si milarity of its ESR signal with that of single nitrogen-centered monoc ation radical (N-N+). EDTA degradation by oxidative decarboxylation du e to two-electron oxidation from the same or both nitrogen atoms is no t evident, and EDTA concentration remains the same throughout the reac tions. While EDTA binds (K-D = 15 mM) at or near the iodide binding si te (Bhattachsaryya et al. (1993) Biochem. J. 289, 575-580), I+ binds t o HRP with a K-D value of 20 +/- 7 mu M. I+ binding in the HRP-CN comp lex(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 ag ainst various pHs shows the involvement of an ionizable group on the e nzyme having pK(a) = 4.8, contributed by an acidic group, deprotonatio n of which favors I+ binding. Systematic variation of the concentratio ns of H2O2, EDTA, and I+ under steady state condition yields sets of k inetic parameters containing both kinetic and mechanistic information. Four distinct enzyme species are involved in I+ reduction: native enz yme, compound I, compound I-I+, and compound II-I . complex; and rate constants- for individual steps are calculated. The kinetic experiment s support the View that an active enzyme-I+-EDTA ternary complex is fo rmed during I+ reduction by EDTA at the enzyme active site.