The need for iodide in biology is almost exclusively limited to its role in thyroid hormones, yet the recycling of thyroidal iodide is still critical for human health. The flavoprotein iodotyrosine deiodinase (IYD) salvages iodide from byproducts (mono– and diiodotyrosine, MIT and DIT) of thyroid hormone biosynthesis. The original proposal for the deiodination mechanism of IYD included a nucleophilic attack on the iodo group by an active site cysteine. Although this proposal had strong precedence, site–directed mutagenesis has now proven this wrong. Further investigation of the IYD mechanism required large scale protein expression and isolation. This was stymied by the lack of a convenient isolation system until a truncated and soluble version of wild–type IYD could be expressed in yeast and insect cells.

Large scale isolation of this soluble enzyme derivative provided the necessary material for crystallographic studies that in turn resulted in a structure of IYD at 2.0 Å resolution. The structure verified IYD's assignment in the NAD(P)H oxidase/flavin reductase superfamily and showed that no cysteine residues were in the active site. Structures of IYD with bound MIT and DIT were also obtained and indicated that these substrates are sequestered within the active site by inducing helical structure in two otherwise disordered regions of the enzyme to form an active site lid. This lid confers substrate specificity and is critical in positioning substrate such that it stacks on the isoalloxazine of the flavin mononucleotide (FMN) cofactor. Further investigation identified 3–bromo and 3–chlorotyrosine as substrates for IYD, while 3–fluorotyrosine was not dehalogenated by IYD. These new substrates illustrate IYD's activity as a general dehalogenase and IYD's strong dehalogenating power. Mechanistic studies utilizing 5–deazaFMN, which is incapable of performing 1 electron processes, indicated that IYD dehalogenation occurs via two sequential 1 electron transfers from reduced FMN to substrate. Anaerobic single turnover assays and mechanistic precedence have led to a likely mechanism of dehalogenation for IYD involving substrate tautomerization followed by injection of an electron into the carbonyl of the keto intermediate which then facilitates dehalogenation.