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++αKG)PHD2 is 6-coordinate, with a 2His/1Asp facial triad occupying 3 coordination sites, a bidentate α-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 O2 to bind at the iron. Herein we show that steady-state turnover is faster under acidic conditions, with kcat exhibiting a kinetic pKa = 7.22. A variety of spectroscopic probes were employed to identify the active-site acid, through comparison of (Fe2++αKG)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; EXAFS analysis found a short Fe-(O/N) bond length of 1.96 Å 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 SIE of on kcat observed (D2Okcat = 0.91 ± 0.03) for the acid form; a similar SIE was observed for the basic form of enzyme (D2Okcat = 0.9 ± 0.1), with an acid equilibrium offset of ΔpKa = 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 O2 binding or activation, and suggesting that aquo release is relatively slow. The unusual kinetic pKa further suggested that PHD2 might function physiologically to sense both intracellular pO2 as well as pH, which could provide for feedback between anaerobic metabolism and hypoxia sensing.
HIF prolyl-4-hydroxylase 2 (PHD2) is a non-heme Fe, 2-oxoglutarate (2OG) dependent dioxygenase that regulates the hypoxia inducible transcription factor (HIF) by hydroxylating two conserved prolyl residues in N-terminal oxygen degradation domain (NODD) and C-terminal oxygen degradation domain (CODD) of HIF-1α. Prior studies have suggested that the substrate preference of PHD2 arises from binding contacts with the β2β3 loop of PHD2. In this study we tested the substrate selectivity of PHD2 by kinetic competition assays, varied ionic strength, and global protein flexibility using amide H/D exchange (HDX). Our results revealed that PHD2 preferred CODD by 20-fold over NODD and that electrostatics influenced this effect. Global HDX monitored by mass spectrometry indicated that binding of Fe(II) and 2OG stabilized the overall protein structure but the saturating concentrations of either NODD or CODD caused an identical change in protein flexibility. These observations imply that both substrates stabilize the β2β3 loop to the same extent. Under unsaturated substrate conditions NODD led to a higher HDX rate than CODD due to its lower binding affinity to PHD2. Our results suggest that loop closure is the dominant contributor to substrate selectivity in PHD2.
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 under limiting O2 is crucial for understanding how these enzymes sense 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 sidechain 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 WT-PHD2, but kcat and kcat/KM(O2) for Thr387→Ala were increased by roughly 15-fold. X-band EPR 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 seen for WT-PHD2, indicating an altered conformation for bound gas in this variant. Here we show that the sidechain of residue Thr387 plays a significant role in determining the rate of turnover by PHD2 at low [O2].
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