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
-2-
quench ⁄MS experiments demonstrate that reaction of a complex of prolyl
hydroxylase domain 2, Fe(II), 2-oxoglutarate and the C-terminal
oxygendependent degradation domain of hypoxia-inducible factor-a 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; Mo¨ ssbauer 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.