EVIDENCE FOR A RADICAL MECHANISM IN PEROXIDASE-CATALYZED COUPLING .2. SINGLE TURNOVER EXPERIMENTS WITH HORSERADISH-PEROXIDASE

Single turnover experiments were performed with horseradish peroxidase (HRP) to study the mechanism of peroxidase-catalyzed coupling and its stimulation by low concentrations of free diiodotyrosine (DIT). HRP was used because, unlike thyroid peroxidase (TPO) and lactoperoxidase (LPO), the spectral properties of compounds I and II are readily distinguishable. This made it possible to correlate the kinetics and stoichiometry of T-4 + T-3 formation with spectral data. Incubation of 2 mu M preformed HRP-I with 2 mu M [I-125]Tg (thyroglobulin of low hormone content, high iodotyrosine content) in the presence of 1 mu M free DIT yielded about 0.8 residue T-4 and 0.2 residue T-3 per molecule of Tg. This represents the theoretical maximum for iodothyronine formation, indicating remarkably efficient use of the oxidizing equivalents in HRP-I for coupling. The time course for formation of T-4 + T-3 was biphasic. During a rapid initial phase (about 1 min), HRP-I was completely converted to HRP-II, coincident with the formation of about 0.65 residues of T-4 + T-3. During the second slower phase, lasting 10-15 min, HRP-II was completely reduced to the native enzyme, with formation of the remaining T-4 + T-3. In the absence of DIT, the coupling yield was reduced to 0.5-0.6 residue T-4 + T-3 per molecule Tg, and the reaction, although considerably slower, was still biphasic. The rapid phase again corresponded to the conversion of HRP-I to HRP-II, and the slower phase to the conversion of HRP-II to native enzyme. To gain insight into the mechanism of the stimulatory effect of free DIT on coupling, we studied the reaction of DIT with HRP-I and HRP-II. Free DIT reacted with both HRP-I and HRP-II in one-electron transfer reactions, and the time course for these reductions resembled those observed with DIT + Tg. These observations suggest that in DIT-stimulated coupling, free DIT radicals act as a shuttle for transferring oxidizing equivalents from the peroxidase intermediates to the DIT residues in Tg. The remarkable efficiency of the HRP-I-mediated coupling reaction implies that (i) only hormonogenic residues in Tg are oxidized and (ii) oxidation of two hormonogenic residues occurs within the same molecule of Tg. A scheme which attempts to explain both kinetic and stoichiometric features of the coupling reaction observed in this study is proposed. This scheme is based on a radical mechanism, consistent with the conclusions reached in the companion paper. It shows that HRP-II is an obligatory intermediate when coupling is initiated with HRP-I. In confirmation of previous studies, we observed in single turnover experiments that cytochrome c peroxidase compound ES and the protein radical form of LPO compound I are also very efficient mediators of coupling in the presence of free DIT. The observation that single turnover coupling with HRP-I is considerably faster than with the protein radical form of LPO-I raises the possibility that, under catalytic conditions, coupling with LPO (and also with TPO) may involve, at least partially, the pi-cation radical form of compound I. (C) 1993 Academic Press, Inc.