Role of a secondary coordination sphere residue in halogenation catalysis of non-heme iron enzymes

Chemo- and regio-selective catalysis of C(sp3)-H halogenation reaction is a formidable goal in chemical synthesis. 2-oxo-glutarate (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 understood. In this work, we show that tyrosine placement in the SCS of non-heme iron halogenases have a huge impact on their structure, function, and reactivity. We discover that a tyrosine mutant (F121Y) in SyrB2 halogenase undergoes post-translational oxidation to dihydroxyphenylalanine (DOPA) physiologically. A combination of spectroscopic, mass-spectrometric, and biochemical studies show that the DOPA modification in SyrB2 renders the enzyme non-functional. Further bioinformatics analysis suggests that halogenases, unlike hydroxylases, have a conserved placement of phenylalanine at position 121 to preclude such unproductive oxidation. Overall, this study demonstrates the importance of the SCS in controlling the structure and enzymatic activity of non-heme iron halogenases. Our results will have significant implications towards the design of small-molecule and protein-based halogenation catalysts.