Prolyl hydroxylase domain 2 (PHD2) is deemed a primary oxygen sensor in humans, yet many details of its underlying mechanism are still not fully understood. (Fe2+ + alpha KG)PHD2 is 6-coordinate, with a 2His/1Asp facial triad occupying three coordination sites, a bidentate alpha-ketoglutarate occupying two sites, and an aquo ligand in the final site. Turnover is thought to be initiated upon release of the aquo ligand, creating a site for O-2 to bind at the iron. Herein we show that steady-state turnover is faster under acidic conditions, with k(cat), exhibiting a kinetic pK(a) = 7.22. A variety of spectroscopic probes were employed to identify the active-site acid, through comparison of (Fe2+ + alpha KG)PHD2 at pH 6.50 with pH 8.50. The near-UV circular dichroism spectrum was virtually unchanged at elevated pH, indicating that the secondary structure did not change as a function of pH. UV-visible and Fe X-ray absorption spectroscopy indicated that the primary coordination sphere of Fe2+ changed upon increasing the pH; extended X-ray absorption fine structure analysis found a short Fe-(O/N) bond length of 1.96 angstrom at pH 8.50, strongly suggesting that the aquo ligand was deprotonated at this pH. Solvent isotope effects were measured during steady-sate turnover over a wide pH-range, with an inverse solvent isotope effect (SIE) of k(cat) observed ((D2O)k(cat)= 0.91 +/- 0.03) for the acid form; a similar SIE was observed for the basic form of the enzyme ((D2O)k(cat) = 0.9 +/- 0.1), with an acid equilibrium offset of Delta pK(a) = 0.67 +/- 0.04. The inverse SIE indicated that aquo release from the active site Fe2+ immediately precedes a rate-limiting step, suggesting that turnover in this enzyme may be partially limited by the rate of O-2 binding or activation, and suggesting that aquo release is relatively slow. The unusual kinetic pK(a) further suggested that PHD2 might function physiologically to sense both intracellular pO(2) as well as pH, which could provide for feedback between anaerobic metabolism and hypoxia sensing.