Steady-state kinetics of FIH variants with CTAD as the varied substrate. WT (■) D201G (●) Inset: D201E (▼) D201A (◆). All assays contained saturating concentrations of FeSO4 (25 μM), αKG (100 μM), and ascorbate (2 mM), with an ambient O2 concentration (217 μM) and were performed in 50 mM HEPES containing 100 mM NaCl, pH 7.00, 37°C.