Thyroid hormone synthesis in thyroglobulin. The mechanism of the coupling reaction

[U-14C]Tyrosine-labeled noniodinated hog thyroglobulin was iodinated enzymatically and nonenzymatically (iodine, iodide-chloramine-T, pH 7.4, or iodine monochloride, pH 8.1). This led to similar levels of iodine incorporation as well as of thyroid hormone synthesis. Iodine monochloride at pH 5.5 formed "hormonogenic" iodotyrosine residues, but no hormone residues. The latter were formed when the iodinated thyroglobulin was brought to pH 8.5 and then treated with horseradish peroxidase and glucose-glucose oxidase in the absence of iodide and iodine monochloride. Enzymatic hydrolysates contained labeled hormone and pyruvic acid; acid hydrolysates labeled thyronine and acetic acid. (Treatment with acid converts hormone to thyronine and pyruvic to acetic acid.) After borohydride treatment, labeled alanine was present instead of pyruvic or acetic acid. The pyruvic acid/hormone, acetic acid/thyronine, alanine/hormone, and alanine/thyronine molar ratios always were 1, independently of the method of iodination. The "coupling reaction" consists of an oxidation step and nonoxidative coupling and decomposition steps. The oxidation step may be either enzymatic or nonenzymatic. The decomposition step always leads to 1 dehydroalanine residue for each hormone residue synthesized. (Dehydroalanine residues appear in the various hydrolysates as acetic acid, pyruvic acid, and alanine, respectively.) Since proper alignment of 2 iodotyrosine residues is a prerequisite for coupling, a model is proposed according to which oxidation of hormonogenic iodotyrosine residues leads to a charge transfer complex which is the same zwitterion-biradical resonance hybrid no matter whether it resulted from a free radical (enzymatic) or an ionic (nonenzymatic) oxidation.