PHD2 is a 2-oxoglutarate, non-heme Fe2+-dependent oxygenase that senses O2 levels in human cells by hydroxylating two prolyl residues in the oxygen-dependent degradation domain (ODD) of HIF1α. Identifying the active site contacts that determine the rate of reaction at limiting O2 concentrations is crucial for understanding how this enzyme senses pO2 and may suggest methods for chemically altering hypoxia responses. A hydrogen bonding network extends from the Fe(II) cofactor through ordered waters to the Thr387 residue in the second coordination sphere. Here we tested the impact of the side chain of Thr387 on the reactivity of PHD2 toward O2 through a combination of point mutagenesis, steady state kinetic experiments and {FeNO}7 EPR spectroscopy. The steady state kinetic parameters for Thr387 → Asn were very similar to those of wild-type (WT) PHD2, but kcat and kcat/KM(O2) for Thr387 → Ala were increased by roughly 15-fold. X-Band electron paramagnetic resonance spectroscopy of the {FeNO}7 centers of the (Fe+NO+2OG) enzyme forms showed the presence of a more rhombic line shape in Thr387 → Ala than in WT PHD2, indicating an altered conformation for bound gas in this variant. Here we show that the side chain of residue Thr387 plays a significant role in determining the rate of turnover by PHD2 at low O2 concentrations.