Electrostatically Regulated Active Site Assembly Governs Reactivity in Nonheme Iron Halogenases

Non-heme iron halogenases (NHFe-Hals) catalyze the direct insertion of a chloride ion at an unactivated carbon position using a high-valent haloferryl intermediate. Despite more than a decade of structural and mechanistic characterization, a rigorous understanding of the entire catalytic cycle of NHFe-Hals and how they facilitate binding, activation, and reactivity with specific substrates and functionalizing anions remains unclear. Here, we focus on understanding binding and active site assembly in freestanding halogenases, BesD and HalB, which directly catalyze the chlorination of lysine without the need for a partner protein. While the lysine and chloride binding affinities to BesD's active sites are extremely weak (K-d values of similar to 50 and 560 mM, respectively), we demonstrate strong positive heterotropic cooperativity (cooperativity constant, alpha similar to 15,500) between the lysine and chloride binding events such that they bind efficiently when simultaneously present at physiologically relevant concentrations. Using a combination of computational and rational protein design studies, we identify a negatively charged residue, E119, in BesD that locks the active site assembly unless both the chloride anion and the positively charged lysine substrate are simultaneously present. Removing this electrostatic lock by mutating E119 to polar/neutral glutamine and alanine residues results in a 6.7- and 14-fold increase in affinity for the chloride anion, respectively. A concomitant order of magnitude decrease in chlorination yields is observed as lysine binding is impaired in these mutants, yet the chemoselectivity profile remains rather similar. Beyond such implications for the overall catalytic performance, we show that the electrostatically regulated active site assembly stage of BesD's catalytic cycle can also determine its promiscuity for C-H functionalization with other anions such as bromide, azide, and nitrite. Overall, our studies highlight complex electrostatic effects at play during the active site assembly stage of charged substrates like lysine along with their implications for C-H functionalization performance in BesD-like halogenases.