Electrostatically Regulated Active Site Assembly Governs Reactivity in Nonheme Iron Halogenases

Abstract

Non-heme iron halogenases (NHFe-Hals) catalyze the direct insertion of a chloride ion at an unactivated carbon position using a highvalent 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 ∼50 and 560 mM, respectively), we demonstrate strong positive heterotropic cooperativity (cooperativity constant, α ∼ 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.

Controllable multi-halogenation of a non-native substrate by SyrB2 iron halogenase

“…Thus far, we’ve demonstrated control over the degree of multi-chlorination, however, iron halogenases are also capable of mono-brominating their native substrates. 14,16,23,59,71 Given the propensity for the SyrB1-Ppt-Aba substrate to form multi-chlorinated products, next, we investigated if this reactivity could be extended to multi-bromination. We note that multi-bromination reactions are harder to accomplish as H-abstraction, ferryl decay and radical rebound are all rendered slower for bromination as compared to chlorination.…”

Equilibrium dialysis with HPLC detection to measure substrate binding affinity of a non-heme iron halogenase

“…Using equilibrium dialysis methods for lysine binding to BesD, we were able to determine apparent K d for lysine as 43 ± 3 μM and 170 ± 20 μM at chloride concentrations of 30 mM and 5 mM respectively (Fig 3B ) (Smithwick et al, 2023). In the absence of any chloride, these equilibrium dialysis measurements found negligible binding of lysine to BesD which matched with our observations from UV-Vis spectral shift studies.…”

“…(B) HPLC traces from AQC-derivatized standard lysine with calibration curve relating relevant peak area to sample concentration shown as an inset. Reprinted with permission from (Smithwick et al, 2023). Copyright 2023 American Chemical Society.…”

“…(B) Equilibrium dialysis data and derived K d,app for lysine binding to BesD at different concentrations of chloride. Reprinted with permission from (Smithwick et al, 2023). Copyright 2023 American Chemical Society.…”

“…A variety of methods have been used to determine substrate affinity in these and related classes of enzymes, including tryptophan fluorescence (Martin et al, 2019), SPR (Hu et al, 2015), and UV-Vis spectral shift-based methods (Chan et al, 2022; Matthews et al, 2014; Price et al, 2003; Ryle et al, 1999) to name a few. During our investigations of BesD, a non-heme iron halogenase that natively chlorinates lysine at the γ-position (Marchand et al, 2019), we characterized a positive heterotropic cooperativity that exists between the primary substrate lysine and the co-substrate chloride (Smithwick et al, 2023). To quantify how the concentration of lysine influences the binding of chloride, we were able to utilize a UV-Vis spectral shift-based assay which takes advantage of a perturbation that occurs in a metal-to-ligand charge transfer (MLCT) band between the ferrous iron center and the co-substrate 2-oxoglutarate (2OG) upon the direct binding of chloride to iron (Matthews et al, 2014).…”