Role of Secondary Coordination Sphere Residues in Halogenation Catalysis of Non-heme Iron Enzymes

Abstract

Chemo- and regio-selective catalysis of the C(sp3)-H halogenation reaction is a formidable goal in chemical synthesis. 2-Oxoglutarate (2OG)-dependent non-heme iron halogenases catalyze selective chlorination/bromination of C–H bonds and exhibit high sequence and structural similarities with non-heme iron hydroxylases. How the secondary coordination sphere (SCS) of these two enzyme systems differentiate and determine their reactivity is not well understood. In this work, we show that specific positioning of redox-active tyrosine residues in the SCS of non-heme iron halogenases has a huge impact on their structure, function, and reactivity. We discover that a tyrosine residue (F121Y) rationally incorporated to hydrogen bond to iron’s chloride ligand in SyrB2 halogenase undergoes post-translational oxidation to dihydroxyphenylalanine (DOPA) physiologically. A combination of spectroscopic, mass-spectrometric, and biochemical studies demonstrate that DOPA modification in SyrB2 renders the enzyme non-functional. Bioinformatic analysis suggests that SyrB2-like halogenases, unlike hydroxylases, have a conserved placement of phenylalanine at position 121 to preclude such unproductive oxidation. Furthermore, molecular dynamics simulations in tandem with experimental demonstration of DOPA incorporation exclusively at position 121 enables us to uniquely identify that an axial-chloro haloferryl isomer is operant in SyrB2. We also identify conserved redox-inactive residues in the SCS of other 2OG-dependent non-heme iron halogenases to avoid DOPA-like unproductive oxidations. Overall, this study demonstrates the importance of the SCS in controlling the structure and enzymatic activity of non-heme iron halogenases and will have significant implications toward the design of small-molecule and protein-based halogenation catalysts.