Substrate positioning controls the partition between halogenation and hydroxylation in the aliphatic halogenase,...

The alpha-ketoglutarate-dependent hydroxylases and halogenases employ similar reaction mechanisms involving hydrogen-abstracting Fe(IV)-oxo (ferryl) intermediates. In the halogenases, the carboxylate residue from the His(2)(Asp/Glu)(1) "facial triad" of iron ligands found in the hydroxylases is replaced by alanine, and a halide ion (X(-)) coordinates at the vacated site. Halogenation is thought to result from "rebound" of the halogen radical from the X-Fe(III)-OH intermediate produced by hydrogen (H(*)) abstraction to the substrate radical. The alternative decay pathway for the X-Fe(III)-OH intermediate, rebound of the hydroxyl radical to the substrate radical (as occurs in the hydroxylases), reportedly does not compete. Here we show for the halogenase SyrB2 that positioning of the alkyl group of the substrate away from the oxo/hydroxo ligand and closer to the halogen ligand sacrifices H(*)-abstraction proficiency for halogen-rebound selectivity. Upon replacement of L-Thr, the C4 amino acid tethered to the SyrB1 carrier protein in the native substrate, by the C5 amino acid L-norvaline, decay of the chloroferryl intermediate becomes 130x faster and the reaction outcome switches to primarily hydroxylation of C5, consistent with projection of the methyl group closer to the oxo/hydroxo by the longer side chain. Competing H(*) abstraction from C4 results primarily in chlorination, as occurs at this site in the native substrate. Consequently, deuteration of C5, which slows attack at this site, switches both the regioselectivity from C5 to C4 and the chemoselectivity from hydroxylation to chlorination. Thus, substrate-intermediate disposition and the carboxylate --> halide ligand swap combine to specify the halogenation outcome.