Two interconverting Fe(IV) intermediates in aliphatic chlorination by the halogenase CytC3

Enzymatic incorporation of a halogen atom is a common feature in the biosyntheses of more than 4,500 natural products(1-5). Halogenation of unactivated carbon centers in the biosyntheses of several compounds of nonribosomal peptide origin is carried out by a class of mononuclear nonheme iron enzymes that require alpha-ketoglutarate ( alpha KG, 1), chloride and oxygen(6). To investigate the ability of these enzymes to functionalize unactivated methyl groups, we characterized the chlorination of the gamma-methyl substituent of L-2-aminobutyric acid ( L-Aba, 2) attached to the carrier protein CytC2 by iron halogenase ( CytC3) from soil Streptomyces sp. We identified an intermediate state comprising two high-spin Fe(IV) complexes in rapid equilibrium. At least one of the Fe( IV) complexes abstracts hydrogen from the substrate. The demonstration that chlorination proceeds through an Fe( IV) intermediate that cleaves a C-H bond reveals the mechanistic similarity of aliphatic halogenases to the iron- and alpha KG- dependent hydroxylases.