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 non-gaseous substrates must bind to activate the enzyme’s Fe(II) cofactor for efficient capture of O2. Halide, 2OG, and (lastly) O2all coordinate directly to the cofactor to initiate its conversion to acis-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 thel-lysine 4-chlorinase, BesD. After 2OG adds, subsequent coordination of the halide to the cofactor and binding of cationicl-Lys near the cofactor are associated with strong heterotropic cooperativity. Progression to the haloferryl intermediate upon addition of O2does not trap the substrates in the active site and, in fact, markedly diminishes cooperativity between halide andl-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 inl-Lys chlorination, especially at low chloride concentrations; one identified pathway involves oxidation of glycerol. The mechanistic data imply that (i) 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 betweenl-Lys binding and chloride coordination following loss of the anionic protein-carboxylate iron ligand present in extant hydroxylases.