The catalytic versatility of flavins encompasses the ability to catalyze one and two electron redox processes as well as dioxygen activation. In flavoenzymes, protein-cofactor interactions modulate flavin chemistry enabling these enzymes to perform a diverse set of biological roles. Flavin analogs, such as deoxyflavins, provide a convenient method for identifying which polar contacts to FMN are necessary for enzymatic activity.

Iodotyrosine deiodinase (IYD) is responsible for the deiodination of byproducts from thyroxine biosynthesis (mono- and diiodotyrosine (MIT and DIT)) in the thyroid. A unique flavoenzyme, IYD, is one of few known aerobic enzymes able to catalysis reductive dehalogenation. Literature precedence for the deiodination mechanism of IYD has not been found. Detection of the flavin semiquinone (Flsq) intermediate during anaerobic reduction of IYD in the presence of substrate suggests deiodination occurs via two single electron transfer processes. Proposed one electron mechanisms for IYD involving formation of a substrate keto-tautomer are supported by the enzyme’s preference for the phenolate form of substrate during binding. Previous analysis of a co-crystal of IYD and MIT showed the involvement of substrate in extensive interactions with the FMN cofactor, including a hydrogen bond between the FMN ribityl 2ʹ-OH and the phenolic OH of MIT. This hydrogen bond has been hypothesized to activate substrate for deiodination.

To probe the significance of the polar interaction between the ribityl 2ʹ-OH and the phenolic OH of MIT in IYD, 2-deoxyriboflavin was synthesized and enzymatically phosphorylated using riboflavin kinase. Reconstitution of human IYD (hIYD) with 2-deoxyFMN produced an enzyme with a significantly decreased affinity for MIT. Deiodinase activity was retained in 2-deoxyhIYD with a 10-fold decrease in catalytic efficiency. Detection of the Flsq was not observed during anaerobic reduction of 2-deoxyhIYD in the presence of mono-fluorotyrosine (MFT). The enzyme’s preference for the phenol versus phenolate form of substrate could not be determined due to the low solubility of DIT at concentrations necessary for pH dependent binding analysis. Removal of the ribityl 2ʹ-OH did not support turnover of O-methyl MIT, a substrate analog incapable of undergoing tautomerization.