MECHANISM OF SIMULTANEOUS IODINATION AND COUPLING CATALYZED BY THYROID PEROXIDASE

Thyroid peroxidase (TPO) simultaneously catalyzes two very different t ypes of reaction in the thyroid gland-iodination and coupling, The pre sent study addresses the mechanism of this simultaneous dual activity, Compound I, the two-electron oxidation product of TPO, exists in two different forms-an oxoferryl porphyrin pi-cation radical and an oxofer ryl protein radical, It has been proposed that iodination is mediated by the porphyrin pi-cation radical form of TPO compound I, while coupl ing is mediated by the protein radical form, However, results obtained in the present study favor the view that both iodination and coupling are mediated by the porphyrin pi-cation radical form of compound I, I n the first part of the study, we compared coupling and iodination act ivities of two peroxidases with very similar crystal structures-cytoch rome c peroxidase (CcP) and lignin peroxidase (LiP), Although these tw o peroxidases have very similar three-dimensional structures, CcP form s a compound I only of the protein radical type, whereas compound I of LiP exists only as a porphyrin pi-cation radical, Comparison of the c atalytic activities of the two enzymes showed that diiodotyrosine (DIT )-stimulated coupling activity of LiP was significantly greater than t hat of CcP, Moreover, lignin peroxidase displayed very significant iod inating activity at acid pHs, whereas iodination with CcP was negligib le at all pHs tested, Our findings with these two structurally similar peroxidases suggested that TPO-catalyzed iodination and coupling coul d both be mediated by the porphyrin pi-cation radical form of compound I, More direct evidence in support of this view was obtained in the s econd part of this study, employing TPO and lactoperoxidase (LPO) mode l systems in which iodination and coupling occurred simultaneously, He me spectral analysis was used to correlate formation of the protein ra dical form of compound I with the kinetics of the iodination and coupl ing reactions, Formation of the compound I protein radical was not obs erved until the iodination and coupling reactions had almost been comp leted, In separate experiments it was shown that the spontaneous conve rsion of the porphyrin pi-cation radical form of TPO or LPO compound I to the protein radical form was markedly inhibited by a low concentra tion of iodide, especially in the presence of an iodide acceptor, Thes e studies provide compelling evidence that both iodination and couplin g are mediated by the porphyrin pi-cation radical form of compound I, This was further substantiated by the finding that coupling was inhibi ted in the presence of excess iodide, an observation readily explained by competition between iodide and DIT residues in thyroglobulin for o xidation by the porphyrin pi-cation radical. (C) 1996 Academic Press, Inc.