Abstract
α-Ketoglutarate (αKG) dependent oxygenases comprise a large superfamily of enzymes that activate O(2) for varied reactions. While most of these enzymes contain a nonheme Fe bound by a His(2)(Asp/Glu) facial triad, a small number of αKG-dependent halogenases require only the two His ligands to bind Fe and activate O(2). The enzyme "factor inhibiting HIF" (FIH) contains a His(2)Asp facial triad and selectively hydroxylates polypeptides; however, removal of the Asp ligand in the Asp201→Gly variant leads to a highly active enzyme, seemingly without a complete facial triad. Herein, we report on the formation of an Fe-Cl cofactor structure for the Asp201→Gly FIH variant using X-ray absorption spectroscopy (XAS), which provides insight into the structure of the His(2)Cl facial triad found in halogenases. The Asp201→Gly variant supports anion dependent peptide hydroxylation, demonstrating the requirement for a complete His(2)X facial triad to support O(2) reactivity. Our results indicated that exogenous ligand binding to form a complete His(2)X facial triad was essential for O(2) activation and provides a structural model for the His(2)Cl-bound nonheme Fe found in halogenases.