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

Abstract

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, parahydroxybenzoate hydroxylase. These calculations should guide future enzyme and substrate design strategies and broaden the scope of biological spin chemistry.

Tuning the Quantum Chemical Properties of Flavins via Modification at C8

“…Flavins are present in over 100 different enzymes, usually in the form of either flavin mononucleotide (FMN) or flavin adenine dinucleotide (FAD), but covalently modified in some 10% of cases. , Flavins are at the catalytic or conformational core of dehydrogenases, , oxidases, , oxygenases, electron carriers, , hydrolases, − light-responsive sensors, − light-driven DNA repair, , fuel-producing decarboxylases, dehalogenases, and even magnetosensory navigational systems . Given the extreme versatility of flavins, the second marvel is how individual proteins manage to emphasize a specific activity.…”

New frontiers in flavin-dependent monooxygenases

“…A more recent study also used DFT simulations to investigate the mechanism of substrate hydroxylation in the fungal enzyme TropB, a class A flavin-dependent hydroxylase [61]. It was established that in the reaction catalyzed by TropB, a hydroxyl radical-coupled electron transfer mechanism (HRC-ET) is favored by ~ 7 kcal/mol compared to the electrophilic aromatic substitution.…”

Photocatalytic Oxidative Dearomatization of Orcinaldehyde Derivatives

“…Nature employs flavin-dependent monooxygenases (FDMOs) to perform this challenging oxidation with perfect site- and stereoselectivity . These enzymes use the noncovalent cofactor FADH 2 (see 6 ) which reacts with molecular oxygen to form hydroperoxyflavin ( 7 ), an electrophilic source of oxygen that can react with electron-rich arenes posed in the enzyme active site (Figure C). , This environmentally benign system employs molecular oxygen as the stoichiometric oxidant and water as the solvent, ultimately affording a single product isomer. Recently, we disclosed our computational findings which support a triplet transition state in the reaction between hydroperoxyflavin (see 7 ) and resorcinol 11 .…”

“…Taking advantage of the solvent effect offered by moving to aqueous reaction conditions, wherein the p K a of resorcinol 11 was measured to be 7.2, the phenolate could be generated cleanly under mild conditions. We were gratified to find that in a variety of buffers at a pH of 8.0, with 0.4 mol % FMN, 11 was converted to a single, dearomatized product (Table , entries 4, 6, and 7).…”