Electrostatically regulated active site assembly governs reactivity in non-heme iron halogenases

Non-heme iron halogenases (NHFe-Hals) catalyze the direct insertion of a chloride/bromide ion at an unactivated carbon position using a high-valent haloferryl intermediate. Despite more than a decade of structural and mechanistic characterization, how NHFe-Hals preferentially bind specific anions and substrates for C-H functionalization remains unknown. Herein, using lysine halogenating BesD and HalB enzymes as model systems, we demonstrate strong positive cooperativity between anion and substrate binding to the catalytic pocket. Detailed computational investigations indicate that a negatively charged glutamate hydrogen-bonded to iron's equatorial-aqua ligand acts as an electrostatic lock preventing both lysine and anion binding in the absence of the other. Using a combination of UV-Vis spectroscopy, binding affinity studies, stopped-flow kinetics investigations, and biochemical assays, we explore the implication of such active site assembly towards chlorination, bromination, and azidation reactivities. Overall, our work highlights previously unknown features regarding how anion-substrate pair binding govern reactivity of iron halogenases that are crucial for engineering next-generation C-H functionalization biocatalysts.