Synergistic Binding of the Halide and Cationic Prime Substrate of <scp>l</scp> -Lysine 4-Chlorinase, BesD, in Both Ferrous and Ferryl States

Abstract

An aliphatic halogenase requires four substrates: 2-oxoglutarate (2OG), halide (Cl^- or Br^-), the halogenation target ("prime substrate"), and dioxygen. In well-studied cases, the three nongaseous substrates must bind to activate the enzyme's Fe(II) cofactor for efficient capture of O₂. Halide, 2OG, and (lastly) O₂ all coordinate directly to the cofactor to initiate its conversion to a <i>cis</i>-halo-oxo-iron(IV) (haloferryl) complex, which abstracts hydrogen (H^•) from the non-coordinating prime substrate to enable radicaloid carbon-halogen coupling. We dissected the kinetic pathway and thermodynamic linkage in binding of the first three substrates of the l-lysine 4-chlorinase, BesD. After addition of 2OG, subsequent coordination of the halide to the cofactor and binding of cationic l-Lys near the cofactor are associated with strong heterotropic cooperativity. Progression to the haloferryl intermediate upon the addition of O₂ does not trap the substrates in the active site and, in fact, markedly diminishes cooperativity between halide and l-Lys. The surprising lability of the BesD•[Fe(IV)=O]•Cl•succinate•l-Lys complex engenders pathways for decay of the haloferryl intermediate that do not result in l-Lys chlorination, especially at low chloride concentrations; one identified pathway involves oxidation of glycerol. The mechanistic data imply (i) that BesD may have evolved from a hydroxylase ancestor either relatively recently or under weak selective pressure for efficient chlorination and (ii) that acquisition of its activity may have involved the emergence of linkage between l-Lys binding and chloride coordination following the loss of the anionic protein-carboxylate iron ligand present in extant hydroxylases.