Hydroxyl Radical-Coupled Electron-Transfer Mechanism of Flavin-Dependent Hydroxylases

Class A flavin-dependent hydroxylases (FdHs) catalyze the hydroxylation of organic compounds in a site- and stereoselective manner. In stark contrast, conventional synthetic routes require environmentally hazardous reagents and give modest yields. Thus, understanding the detailed mechanism of this class of enzymes is essential to their rational manipulation for applications in green chemistry and pharmaceutical production. Both electrophilic substitution and radical intermediate mechanisms have been proposed as interpretations of FdH hydroxylation rates and optical spectra. While radical mechanistic steps are often difficult to examine directly, modern quantum chemistry calculations combined with statistical mechanical approaches can yield detailed mechanistic models providing insights that can be used to differentiate reaction pathways. In the current work, we report quantum mechanical/molecular mechanical (QM/MM) calculations on the fungal TropB enzyme that shows an alternative reaction pathway in which hydroxylation through a hydroxyl radical-coupled electron-transfer mechanism is significantly favored over electrophilic substitution. Furthermore, QM/MM calculations on several modified flavins provide a more consistent interpretation of the experimental trends in the reaction rates seen experimentally for a related enzyme, para-hydroxybenzoate hydroxylase. These calculations should guide future enzyme and substrate design strategies and broaden the scope of biological spin chemistry.