Evidence for the slow reaction of hypoxia-inducible factor prolyl hydroxylase 2 with oxygen.
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.
Flashman
Emily
E
Department of Chemistry and Oxford Centre for Integrative Systems Biology, Oxford, UK.
Hoffart
Lee M
LM
Hamed
Refaat B
RB
Bollinger
J Martin
JM
Krebs
Carsten
C
Schofield
Christopher J
CJ
D000595:Amino Acid Sequence; D002384:Catalysis; D006801:Humans; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D010455:Peptides / Q000378:metabolism; D011393:Procollagen-Proline Dioxygenase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D015204:Spectroscopy, Mossbauer; D013379:Substrate Specificity
C497442:HIF1A protein, human; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D010455:Peptides; D011994:Recombinant Proteins; D011393:Procollagen-Proline Dioxygenase