Dynamics and Mechanism of Photoenzymatic Dehalogenation Reactions through Electron-Transfer Bifurcation

Light-induced dehalogenation by flavoenzymes offers a promising route to generate acyl radicals in photoenzymatic catalysis, but current applications are largely limited to α-acyl halides. Mechanistic studies are needed to understand this substituent position effect to engineer flavoenzymes for broader applications. Here, we elucidate the ultrafast dynamics of photoinduced electron transfer (ET) and dehalogenation reactions in lactate monooxygenase (LMO) with α-, β-, and γ-acyl halides. We found that the ET from the excited reduced flavin cofactor (FMNH^-*) to the substrates bifurcates into two pathways: direct tunneling to the C-X (X = Cl, Br) bond or direct hopping to the neighboring carbonyl group. The former leads to instantaneous dehalogenation; the latter forms an anionic (C-O^-) radical that can either transfer the electron to the halogen for dehalogenation or undergo nonproductive back ET (BET). This finding contrasts with conventional understandings that the direct tunneling ET to the halogen atom is the only reaction channel. For α-halogenated substrates, both pathways lead to a dehalogenation reaction. For β-halogenated ones, direct tunneling causes effective dehalogenation and direct hopping leads to a futile BET. For γ-halogenated ones, direct tunneling is negligible and direct hopping results in BET. After dehalogenation, the resulting acyl radical bonds with flavin semiquinone (FMNH^•) to form the photoproduct. Molecular simulations and docking indicate that these outcomes are governed by substrate orientation, structural configuration, and hydrogen-bonding networks. This bifurcating ET mechanism explains the substituent position effects on reactivity and provides a framework for engineering flavoenzymes for dehalogenative reactions.