Substrate Electronics Dominate the Rate of Reductive Dehalogenation Promoted by the Flavin-Dependent Iodotyrosine...

Iodotyrosine deiodinase (IYD) is unusual in its reliance on flavin to promote reductive dehalogenation of halotyrosines under aerobic conditions. Applications of this activity can be envisioned for bioremediation, but expansion of its specificity requires an understanding of the mechanistic steps that limit the rate of turnover. Key processes capable of controlling steady-state turnover have now been evaluated and described in this study. While proton transfer is necessary for converting the electron-rich substrate into an electrophilic intermediate suitable for reduction, kinetic solvent deuterium isotope effects suggest that this process does not contribute to the overall efficiency of catalysis under neutral conditions. Similarly, reconstituting IYD with flavin analogues demonstrates that a change in reduction potential by as much as 132 mV affects k_cat by less than 3-fold. Furthermore, k_cat/K_m does not correlate with reduction potential and indicates that electron transfer is also not rate determining. Catalytic efficiency is most sensitive to the electronic nature of its substrates. Electron-donating substituents on the ortho position of iodotyrosine stimulate catalysis and conversely electron-withdrawing substituents suppress catalysis. Effects on k_cat and k_cat/K_m range from 22- to 100-fold and fit a linear free-energy correlation with a ρ ranging from -2.1 to -2.8 for human and bacterial IYD. These values are consistent with a rate-determining process of stabilizing the electrophilic and nonaromatic intermediate poised for reduction. Future engineering can now focus on efforts to stabilize this electrophilic intermediate over a broad series of phenolic substrates that are targeted for removal from our environment.