Selective Chlorination of Substrates by the Halogenase SyrB2 Is Controlled by the Protein According to a Combined Quantum Mechanics/Molecular Mechanics and Molecular Dynamics Study

The enzyme SyrB2 employs an Fe^IV–oxo species to achieve selective C–H halogenation of l-threonine. Herein, we use combined quantum mechanical/molecular mechanical (QM/MM) calculations and molecular dynamics (MD) simulations to decipher the mechanism of selective halogenation by SyrB2. Our QM/MM calculations show the presence of three Cl–Fe^IV–oxo isomers which interconvert, and only the one having its oxo ligand pointing toward the target C–H bond is active during the hydrogen atom abstraction (H-abstraction) process. The fate of the formed Cl–Fe^III–OH/R^• intermediate is determined by a hydrogen-bonding interaction between the Arg254 residue and the OH ligand of Cl–Fe^III–OH. The hydrogen bond not only prevents the OH group from participating in the followup rebound step to form a hydroxylated product but also facilitates the isomerization of the Cl–Fe^III–OH/R^• intermediate so that the Cl is directed toward the alkyl radical. The role of Arg254 in regulating the selectivity of chlorination is further discussed and connected to the experimentally observed effect of mutations of Arg247 (Arg247Glu and Arg247Ala) in the related CurA halogenase. The Ala118Asp and Ala118Glu mutants of SyrB2 were investigated by MD simulations, and they were found to suppress the H-bonding interaction of Arg254 with Cl–Fe^III–OH: this result is in accord with their experimentally observed suppressed chlorination activity. This novel mechanism highlights the role of the H-bonding interaction between the protein and a reaction intermediate.