FIH [factor inhibiting HIF (hypoxia inducible factor)] is an alpha-ketoglutarate (alpha KG)-dependent nonheme iron enzyme that catalyzes the hydroxylation of the C-terminal transactivation domain (CAD) asparagine residue in HIF-1 alpha to regulate cellular oxygen levels. The role of the facial triad carboxylate ligand in O-2 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 (Fe-II)FIH variants were all 6-coordinate (6C) and the alpha KG plus CAD bound FIH variants were all 5-coordinate (SC), mirroring the behavior of the wild-type (wt) enzyme. When only aKG is bound, all FIH variants exhibited weaker Fe-II-OH2 bonds for the sixth ligand compared to wt, and for alpha KG-bound D201E this is either extremely weak or the site is SC, demonstrating that the Asp201 residue plays an important role in the wt enzyme in ensuring that the (Fe-II/alpha KG)FIH site remains 6C. Variable-temperature, variable-field (VT'VH) MCD spectroscopy showed that all of the alpha KG- and CAD-bound FIH variants, though 5C, have different ground-state geometric and electronic structures, which impair their oxygen activation rates. Comparison of O-2 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.