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

• Published in: The FEBS journal Vol. 277; no. 19; pp. 4089 - 4099
• Main Authors: Flashman, Emily, Hoffart, Lee M, Hamed, Refaat B, Bollinger Jr, J. Martin, Krebs, Carsten, Schofield, Christopher J
• Format: Journal Article
• Language: English
• Published: Oxford, UK Oxford, UK : Blackwell Publishing Ltd 01.10.2010; Blackwell Publishing Ltd
• Subjects: 2-oxoglutarate; Amino Acid Sequence; Catalysis; Humans; Hypoxia; hypoxia-inducible factor; Hypoxia-Inducible Factor 1, alpha Subunit - chemistry; Hypoxia-Inducible Factor 1, alpha Subunit - metabolism; Kinetics; Magnetic Resonance Spectroscopy; Molecular Sequence Data; Oxygen; oxygenase; Peptides - metabolism; procollagen-proline dioxygenase; Procollagen-Proline Dioxygenase - chemistry; Procollagen-Proline Dioxygenase - genetics; Procollagen-Proline Dioxygenase - metabolism; prolyl hydroxylase; Recombinant Proteins - chemistry; Recombinant Proteins - metabolism; spectroscopy; Spectroscopy, Mossbauer; Substrate Specificity
• ISSN: 1742-464X; 1742-4658
• DOI: 10.1111/j.1742-4658.2010.07804.x

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.