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Role of the Facial Triad in Factor Inhibiting HIF (FIH): Ligand Binding, Substrate Selectivity, and Coupling

Alpha-ketoglutarate (αKG) dependent oxygenases comprise a large superfamily of enzymes that activate O2 for varied reactions. While most of these enzymes contain a non-heme Fe bound by a His2Asp facial triad, a small number of αKG-dependent halogenases require only the two His ligands to bind Fe and activate O2. The enzyme “factor inhibiting HIF” (FIH) contains a His2Asp facial triad and selectively hydroxylates polypeptides, however removal of the Asp ligand in the D201G 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 D201G variant using x-ray absorption spectroscopy (XAS), which provides insight into the structure of the His2Cl facial triad found in halogenases. D201G variant supports anion dependent peptide hydroxylation, demonstrating the requirement for a complete His2X facial triad to support O2 reactivity. Our results indicate that exogenous ligand binding to form a complete His2X facial triad was essential for O2 activation, and provides a structural model for the His2Cl-bound nonheme Fe found in halogenases. We also propose that the facial triad ligands couple O2 activation with hydroxylation in FIH, providing insight into the coupling mechanism for this broad class of enzymes. Mutating the Asp201 ligand resulted in uncoupled product formation. For the D201X variants, O2 was consumed much faster than primary substrate was hydroxylated, which suggested branching in the chemical mechanism. EPR studies established that this branching occurs following the formation of the Fe(IV)=O intermediate as the metal centers of WT FIH and D201G were triggered by substrate whereas the metal centers of D201E and D201A were unaffected. Investigation into the fate of O2 in the absence of primary substrate identified two products: hydrogen peroxide (H2O2) and autohydroxylated Trp (TrpOH). This study provides insight into significance of the facial triad in substrate triggering within the αKG oxygenase family of enzymes and describes the products of uncoupling.
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