Evidence for the slow reaction of hypoxia-inducible factor prolyl hydroxylase 2 with oxygen

Abstract

The response of animals to hypoxia is mediated by the hypoxia-inducible transcription factor. Human hypoxia-inducible factor is regulated by four Fe(II)- and 2-oxoglutarate-dependent oxygenases: prolyl hydroxylase domain enzymes 1-3 catalyse hydroxylation of two prolyl-residues in hypoxia-inducible factor, triggering its degradation by the proteasome. Factor inhibiting hypoxia-inducible factor catalyses the hydroxylation of an asparagine-residue in hypoxia-inducible factor, inhibiting its transcriptional activity. Collectively, the hypoxia-inducible factor hydroxylases negatively regulate hypoxia-inducible factor in response to increasing oxygen concentration. Prolyl hydroxylase domain 2 is the most important oxygen sensor in human cells; however, the underlying kinetic basis of the oxygen-sensing function of prolyl hydroxylase domain 2 is unclear. We report analyses of the reaction of prolyl hydroxylase domain 2 with oxygen. Chemical quench/MS experiments demonstrate that reaction of a complex of prolyl hydroxylase domain 2, Fe(II), 2-oxoglutarate and the C-terminal oxygen-dependent degradation domain of hypoxia-inducible factor-α with oxygen to form hydroxylated C-terminal oxygen-dependent degradation domain and succinate is much slower (approximately 100-fold) than for other similarly studied 2-oxoglutarate oxygenases. Stopped flow/UV-visible spectroscopy experiments demonstrate that the reaction produces a relatively stable species absorbing at 320 nm; Mössbauer spectroscopic experiments indicate that this species is likely not a Fe(IV)=O intermediate, as observed for other 2-oxoglutarate oxygenases. Overall, the results obtained suggest that, at least compared to other studied 2-oxoglutarate oxygenases, prolyl hydroxylase domain 2 reacts relatively slowly with oxygen, a property that may be associated with its function as an oxygen sensor.

Figure 1

Proposed general catalytic mechanism for the Fe(II)/2OG oxygenases.

Figure 2

PHD2:Fe(II):2OG:CODD reacts slowly with oxygen in vitro. In the presence of CODD peptide substrate, 2OG decarboxylation to succinate (black circles) and CODD hydroxylation (red circles) occur at similar rates of 0.018s⁻¹ and 0.013s⁻¹ respectively, as determined by LC/MS and MALDI MS respectively. In the absence of CODD peptide substrate, 2OG decarboxylation to succinate (white circles) is 30-fold slower, at 0.0006s⁻¹. Data are shown against time on a logarithmic scale. Concentrations before mixing were PHD2 (0.8 mM), Fe (0.7 mM), 2OG (0.5 mM), ascorbate (5 mM), CODD peptide (1 mM if present) and oxygen (1.9 mM). All reactions were carried out at 5 °C.

Figure 3

¹H-Nuclear magnetic resonance time course demonstrating that 2-oxoglutarate decarboxylation is coupled to conversion to succinate and CODD peptide hydroxylation during reaction of PHD2. A, Full spectra of assay mixtures (see Experimental Details) as measured at 0, 5, 10, 15 and 20 minutes (blue, red, green, violet and yellow respectively). B, Conversion of 2OG to succinate: 2OG was monitored by the triplet at 2.42ppm, and succinate by the singlet at 2.39ppm. C, An increase in the intensity of the ¹H-NMR signal at 3.78ppm, previously assigned as the δ proton of Pro-564 [39]. For clarity in B and C, only spectra recorded every 225s are shown. D, Integrated ¹H-NMR signal intensities for 2OG, succinate and hydroxylated CODD (n=3), showing that 2OG decarboxylation and CODD hydroxylation rates are coupled in steady state turnover experiments. Data were fitted by the equation, y=(y0 − plateau)*exp(−K*X) + plateau, using Prism 5™.

Figure 4

UV-visible absorption spectra on reaction of PHD2:Fe(II):2OG with an equal volume of an oxygen-saturated buffer, with and without CODD peptide substrate. A, Formation of species absorbing at 320, 380 and 520nm with time in the absence of substrate. B, Formation of species absorbing at 320, 380 and 520 over time in the presence of CODD. C, Broad spectral features observed at a range of time points in the presence of CODD. Concentrations before mixing were PHD2 (0.8 mM), Fe (0.7mM), 2OG (10mM), ascorbate (5mM), CODD peptide (1.0mM) and oxygen (1.9mM). Reactions were carried out at 5°C.

Figure 5

4.2-K/zero-field Mössbauer spectra of the PHD2:Fe(II):2OG:CODD complex before (A) and after reaction with an O₂–saturated buffer solution for 200 s (B), and of the PHD2:Fe(II):2OG: complex before (D) and after reaction with an equal volume of an O₂–saturated buffer solution for 200 s (E). Reaction conditions are given in Materials and Methods. (C) is the difference spectrum (B) – (A). The solid lines in (A) and (D) are quadrupole doublet simulations using the parameters described in the text.