Characterization of a flavoprotein iodotyrosine deiodinase from bovine thyroid. Flavin nucleotide binding and oxidation-reduction properties.
A stable apoprotein has been prepared from a soluble purified bovine thyroid iodotyrosine deiodinase, previously shown to be an FMN-containing flavoprotein requiring dithionite for enzymatic activities. The apoprotein binds FMN (Ka = 1.47 x 10(8) M-1) with an almost complete restoration of enzymatic activity. It can also bind FAD (Ka = 0.58 x 10(8) M-1) with partial restoration of activity, but does not bind riboflavin. Photoreduction of the holoenzyme in presence of excess of its free cofactor, FMN, supported enzyme activity at a level of 50% of that obtained with dithionite; substituting FAD or riboflavin for FMN produced, respectively, 20 and 11% of the dithionite-supported activity. The oxidation-reduction potential (E1) of the couple semiquinone/fully reduced enzyme is -0.412 V at pH 7 and 25 degrees C. The value (E2) for the oxidized/semiquinone couple is -0.190 V at pH 7 and 25 degrees C. Potentiometric titrations with sodium hydrosulfite suggests that the enzyme is reduced in two successive 1-electron oxidation-reduction steps. Effects of pH on E1 suggest ionization of the protonated flavin with an ionization constant of 5.7 x 10(-7). The highly negative oxidation-reduction potential for the fully reduced enzyme species and the apparent requirement for full reduction for enzymatic activity suggests that in NADPH-mediated microsomal deiodination an NADPH-linked electron carrier of suitably negative midpoint potential is a probable intermediate.
D000818:Animals; D002417:Cattle; D004227:Dithionite; D005486:Flavin Mononucleotide / Q000032:analysis; D005420:Flavoproteins / Q000378:metabolism*; D007453:Iodide Peroxidase / Q000378:metabolism*; D007700:Kinetics; D008861:Microsomes / Q000201:enzymology; D010084:Oxidation-Reduction; D010544:Peroxidases / Q000378:metabolism*; D011199:Potentiometry; D011485:Protein Binding; D013053:Spectrophotometry; D013961:Thyroid Gland / Q000201:enzymology*
D005420:Flavoproteins; D004227:Dithionite; D005486:Flavin Mononucleotide; D010544:Peroxidases; D007453:Iodide Peroxidase