Chloride Supports O Activation in the D201G Facial Triad Variant of Factor-Inhibiting Hypoxia Inducible Factor, an α-Ketoglutarate Dependent Oxygenase.

α-Ketoglutarate (αKG) dependent oxygenases comprise a large superfamily of enzymes that activate O for varied reactions. While most of these enzymes contain a nonheme Fe bound by a His(Asp/Glu) facial triad, a small number of αKG-dependent halogenases require only the two His ligands to bind Fe and activate O. The enzyme "factor inhibiting HIF" (FIH) contains a HisAsp 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 HisCl facial triad found in halogenases. The Asp201→Gly variant supports anion dependent peptide hydroxylation, demonstrating the requirement for a complete HisX facial triad to support O reactivity. Our results indicated that exogenous ligand binding to form a complete HisX facial triad was essential for O activation and provides a structural model for the HisCl-bound nonheme Fe found in halogenases.