Double Active Species in Flavoenzymes-Catalyzed C–S Bond Cleavage: Synergistic Base-Catalysis by Flavin-N5OOH and Electrophilic Oxidation by Flavin-N5O

Flavoenzymes play a crucial role in biotransformation processes, particularly in the metabolism of sulfur-containing compounds. Despite extensive studies, the mechanism of selective C–S bond cleavage catalyzed by flavoenzymes is not yet fully explored. Here, using extensive molecular dynamics (MD) simulations and QM/MM and QM calculations, our studies reveal that flavin-N5OOH carries out the selective C–H bond deprotonation of the substrate by releasing the strong base of hydroxyl anions (OH – ), which leads to the highly electrophilic flavin-N5O intermediate for the further oxygenation of the substrate carbon anion. The synergistic base-catalysis by flavin-N5OOH and electrophilic oxidation by flavin-N5O enable the C–S bond cleavage of methanesulfonate (MS – ) in MsuD and the desulfurization reaction of N -acetyl- S -alkyl- l -cysteine sulfoxide (NASAC-SO) in CmoJ. The proposed mechanism is consistent with isotope labeling experiments. We assume that such a synergistic mechanism can be applied to other types of C–S bond cleavage catalyzed by flavoenzymes, including SsuD, SfnG, and DmoA. These findings expand our understanding of oxygen activation chemistry in flavoenzymes and highlight the general catalytic mechanism across the superfamily of sulfur-metabolizing flavoenzymes.