Non-heme iron halogenases, such as SyrB2, WelO5, and BesD, halogenate unactivated carbon atoms of diverse substrates at ambient conditions with exquisite selectivity seldom matched by non-biological catalysts. Although crystallography, spectroscopy, and kinetic measurements provide foundational knowledge of enzyme structure and function, critical gaps remain in our understanding of how the protein environment dynamically reorganizes to a catalytically active state capable of halogenation. Using experimentally-guided molecular dynamics (MD) simulations augmented with multi-scale (i.e., QM/MM) simulations of substrate-bound complexes of BesD and WelO5, we investigate substrate/active-site dynamics that enable selective halogenation. Our simulations reveal that active-site configurational isomerization is necessary in WelO5 to attain substrate/active-site geometry consistent with its observed chemo- and regioselectivity. Conversely, a slight reorientation of the substrate from its crystal-structure position is sufficient to enable regioselective chlorination in BesD without the need to invoke active-site isomerization. We observe how substrate-protein interactions evolve during experimentally-motivated MD of halogenases. We relate the nature of these interactions to the distinct substrates. For BesD, we resolve the uncertainty around the mechanistic relevance of Asn219 based on a prior mutagenesis study. By quantifying the presence of thermodynamically competitive C-H bonds on substrates of SyrB2, BesD, and WelO5, we confirm the need for the protein environment to strategically position the substrate to impart regioselectivity to halogenases. Our simulations reveal that the optimum substrate/active-site geometry also outweighs interactions between active site ligands and the protein environment in facilitating the required chemoselectivity in halogenases.
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