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 oxygendependent degradation domain of hypoxia- inducible factor-alpha 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; Mossbauer 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.