The 2'-hydroxy group of flavin mononucleotide influences the catalytic function and promiscuity of the flavoprotein iodotyrosine dehalogenase.

The isoalloxazine ring system of the flavin cofactor is responsible for much of the catalytic power and diversity associated with flavoproteins. While the specificity of these enzymes is greatly influenced by the surrounding protein environment, the ribityl group of the cofactor may also participate in stabilizing transient intermediates formed by substrates and flavin. A conserved interaction between the phenolate oxygen of l-iodotyrosine and the 2'-hydroxy group of flavin mononucleotide (FMN) bound to iodotyrosine deiodianase (IYD) implied such a contribution to catalysis. Reconstitution of this deiodinase with 2'-deoxyflavin mononucleotide (2'-deoxyFMN) decreased the overall catalytic efficiency of l-iodotyrosine dehalogenation (/) by more than 5-fold but increased by over 2-fold. These affects are common to human IYD and its homolog from and are best explained by an ability of the 2'-hydroxy group of FMN to stabilize association of the substrate in its phenolate form. Loss of this 2'-hydroxy group did not substantially affect the formation of the one electron reduced semiquinone form of FMN but its absence released constraints that otherwise suppresses the ability of IYD to promote hydride transfer as measured by a competing nitroreductase activity. Generation of IYD containing 2'-deoxyFMN also removed steric constraints that had previously limited the use of certain mechanistic probes. For example, l--methyl iodotyrosine could be accommodated in the active site lacking the 2'-hydroxy of FMN and shown to be inert to dehalogenation as predicted from a mechanism requiring ketonization of the phenolic oxygen. In the future, ancillary sites within a cofactor should now be considered when engineering new functions within existing protein architectures as demonstrated by the ability of IYD to promote nitroreduction after loss of the 2'-hydroxy group of FMN.