O₂ Activation by Nonheme Fe^II α‑Ketoglutarate-Dependent Enzyme Variants: Elucidating the Role of the Facial Triad Carboxylate in FIH

FIH [factor inhibiting HIF (hypoxia inducible factor)] is an α-ketoglutarate (αKG)-dependent nonheme iron enzyme that catalyzes the hydroxylation of the C-terminal transactivation domain (CAD) asparagine residue in HIF-1α to regulate cellular oxygen levels. The role of the facial triad carboxylate ligand in O2 activation and catalysis was evaluated by replacing the Asp201 residue with Gly (D201G), Ala (D201A), and Glu (D201E). Magnetic circular dichroism (MCD) spectroscopy showed that the (FeII)­FIH variants were all 6-coordinate (6C) and the αKG plus CAD bound FIH variants were all 5-coordinate (5C), mirroring the behavior of the wild-type (wt) enzyme. When only αKG is bound, all FIH variants exhibited weaker FeII–OH2 bonds for the sixth ligand compared to wt, and for αKG-bound D201E this is either extremely weak or the site is 5C, demonstrating that the Asp201 residue plays an important role in the wt enzyme in ensuring that the (FeII/αKG)­FIH site remains 6C. Variable-temperature, variable-field (VTVH) MCD spectroscopy showed that all of the αKG- and CAD-bound FIH variants, though 5C, have different ground-state geometric and electronic structures, which impair their oxygen activation rates. Comparison of O2 consumption to substrate hydroxylation kinetics revealed uncoupling between the two half reactions in the variants. Thus, the Asp201 residue also ensures fidelity between CAD substrate binding and oxygen activation, enabling tightly coupled turnover