The Rate-Limiting Step of O2 Activation in the α-Ketoglutarate Oxygenase Factor Inhibiting Hypoxia Inducible Factor

Factor inhibiting HIF (FIH) is a cellular O2sensing enzyme, which hydroxylates the hypoxia inducible factor-1α. Previously reported inverse solvent kinetic isotope effects indicated that FIH limits its overall turnover through an O2 activation step (Hangasky, J. A., Saban, E., and Knapp, M. J. (2013) Biochemistry 52, 1594−1602). Here we characterize the rate-limiting step for O2 activation by FIH using a suite of mechanistic probes on the second order rate constant kcat/ KM(O2). Steady-state kinetics showed that the rate constant for O2 activation was slow (kcat/KM(O2) app = 3500 M−1 s−1) compared with other non-heme iron oxygenases, and solvent viscosity assays further excluded diffusional encounter with O2 from being rate limiting on kcat/KM(O2). Competitive oxygen-18 kinetic isotope effect measurements ( kcat/KM(O2) = 1.0114(5)) indicated that the transition state for O2 activation resembled a cyclic peroxohemiketal, which precedes the formation of the ferryl intermediate observed in related enzymes. We interpret this data to indicate that FIH limits its overall activity at the point of the nucleophilic attack of Fe-bound O2  on the C-2 carbon of αKG. Overall, these results show that FIH follows the consensus mechanism for αKG oxygenases, suggesting that FIH may be an ideal enzyme to directly access steps involved in O2 activation among the broad family of αKG oxygenases. M cells respond to decreased cellular pO2 levels through the enzyme-catalyzed reaction of O2 with the hypoxia inducible factor-1α (HIF-1α or HIF). HIF mediates the transcription of hundreds of genes in response to hypoxia with the functions of the gene products ranging from glucose and iron metabolism to cell proliferation and angiogenesis. Factor inhibiting HIF (FIH) is a non-heme Fe(II)/αKG oxygenase that turns-off the transcriptional activity of HIF by hydroxylating the β-carbon of Asn within the C-terminal activation domain (CTAD) of HIF (Scheme 1). Because O2 activation chemistry is central to hypoxia sensing by HIF, identifying the chemical steps involved in O2 activation may point the way to methods for perturbing HIF-controlled gene expression. FIH is proposed to follow the consensus mechanism for Fe(II)/αKG oxygenases (Scheme 1) for which the steps are supported to varying degrees by spectroscopic, computational, and kinetic studies. VTVH MCD methodologies have been used to spectroscopically identify the release of the aquo ligand upon substrate binding to FIH and other Fe(II)/αKG oxygenases including TauD and CAS. O2 is thought to bind as a ferric superoxide at the open coordination site and then attacks the C-2 carbonyl of αKG to ultimately form succinate and a ferryl intermediate. The molecular details following O2 activation, including isolation of the ferryl intermediate and observation of HAT have been characterized in the Fe(II)/ αKG oxygenases TauD and P4H and the related Fe(II)/αKG halogenases CytC3 and SyrB2. In contrast to the steps following ferryl formation, those steps of O2 activation are poorly understood. Computational studies suggest that nucleophilic attack on the C-2 carbonyl of αKG is the rate-limiting step on kcat/KM(O2), with a cyclic peroxohemiketal proposed as the transition state. Although this reaction sequence is supported by pre-steadystate kinetics and an oxygen-18 kinetic isotope effect (O KIE) study of TauD, insight into O2 activation is limited because HAT or product release is rate-limiting in TauD and other wellcharacterized αKG oxygenases. Consequently, O2 activation is too rapid to allow for the identification of any intermediates prior to the ferryl. Recent studies showed that the rate-limiting step for FIH differed from that of other characterized αKG oxygenases. Upon FIH binding to CTAD, there is partial retention of the aquo ligand suggesting that aquo release may be less facile in FIH than in other enzymes. The inverse SKIE on kcat 34 for FIH Received: October 2, 2014 Revised: November 20, 2014 Published: November 25, 2014 Article pubs.acs.org/biochemistry © 2014 American Chemical Society 8077 dx.doi.org/10.1021/bi501246v | Biochemistry 2014, 53, 8077−8084 This is an open access article published under an ACS AuthorChoice License, which permits copying and redistribution of the article or any adaptations for non-commercial purposes.