Hypoxia sensing is the generic term for pO 2 -sensing in humans and other higher organisms. These cellular responses to pO 2 are largely controlled by enzymes that belong to the Fe(II) α-ketoglutarate (αKG) dependent dioxygenase superfamily, including the human enzyme called the Factor Inhibiting HIF (FIH-1), which couples O 2 -activation to the hydroxylation of the Hypoxia Inducible Factor α (HIFα). Uncoupled O 2 -activation by human FIH-1 was studied by exposing the resting form of FIH-1, (αKG+Fe)FIH-1, to air in the absence of HIFα. Uncoupling lead to two distinct enzyme oxidations, one a purple chromophore (λ max = 583 nm) arising from enzyme auto-hydroxylation of ; the other a yellow chromophore due to Fe(III) in the active site, which under some conditions also contained variable levels of an oxygenated surface residue, (oxo)Met 275 . The kinetics of purple FIH-1 formation were independent of Fe(II) and αKG concentrations, however product yield was saturable with increasing [αKG] and required excess Fe (II). Yellow FIH-1 was formed from (succinate+Fe)FIH-1, or by glycerol addition to (αKG+Fe) FIH-1, suggesting that glycerol could intercept the active oxidant from the FIH-1 active site and prevent hydroxylation. Both purple and yellow FIH-1 contained high-spin, rhombic Fe(III) centers, as shown by low temperature EPR. XAS indicated distorted octahedral Fe(III) geometries, with subtle differences in inner-shell ligands for yellow and purple FIH-1. EPR of Co(II)-substituted FIH-1, (αKG+Co)FIH-1, indicated a mixture of 5-coordinate and 6-coordinate enzyme forms, suggesting that resting FIH-1 can readily undergo uncoupled O 2 -activation by loss of an H 2 O ligand from the metal center.
The factor inhibiting HIF (FIH) is a proximate oxygen sensor for human cells, hydroxylating Asn803 within the α subunit of the hypoxia inducible factor (HIF). FIH is an α-ketoglutatrate (αKG) dependent, non-heme Fe(II) dioxygenase, in which Fe(II) is coordinated by a (His2Asp) facial triad, αKG, and H2O. Hydrogen bonding between the facial triad, the HIF-Asn803 sidechain, and various second-sphere residues suggests a functional role for the second coordination sphere in tuning the chemistry of the Fe(II) center. Point mutants of FIH were prepared to test the functional role of the αKG-centered (Asn205, Asn294) or HIF-Asn803 centered (Arg238, Gln239) second-sphere residues. The second sphere was tested for local effects on priming Fe(II) to react with O2, oxidative decarboxylation, and substrate positioning. Steady-sate kinetics were used to test for overall catalytic effects, autohydroxylation rates were used to test for priming and positioning, and electronic spectroscopy was used to assess the primary coordination sphere and the electrophilicity of αKG. Asn205→Ala and Asn294→Ala exhibited diminished rates of steady-state turnover, while minimally affecting autohydroxylation, consistent with impaired oxidative decarboxylation. Blue shifted MLCT transitions for (Fe+αKG)FIH indicated that these point mutations destabilized the π* orbitals of αKG, further supporting a slowed rate of oxidative decarboxylation. The Arg238→Met mutant exhibited steady-state rates too low to measure and diminished product yields, suggesting impaired substrate positioning or priming; Arg238→Met was capable of O2-activation for the autohydroxylation reaction. The Gln239→Asn mutant exhibited significantly slowed steady-state kinetics and diminished product yields, suggesting impaired substrate positioning or priming. As HIF binding to Gln239→Asn stimulated autohydroxylation, it is more likely that this point mutant simply mis-positions the HIF-Asn803 sidechain. The present work combines kinetics and spectroscopy to show that these second sphere hydrogen bonds play roles in promoting oxidative decarboxylation, priming Fe(II) to bind O2, and positioning HIF-Asn803.
Oxygen homeostasis plays a critical role in angiogenesis, erythropoiesis and cell metabolism. Oxygen homeostasis is set by the hypoxia inducible factor-1α (HIF-1α) pathway, which is controlled by Factor Inhibiting HIF-1α (FIH). FIH is a non-heme Fe(II), α-ketoglutarate dependent dioxygenase that inhibits HIF-1α by hydroxylating the C-terminal transactivation domain (CTAD) of HIF-1α at HIF-Asn803. A tight coupling between CTAD binding and O2-activation is essential for hypoxia sensing, making changes in the coordination geometry of Fe(II) upon CTAD encounter a crucial feature of this enzyme. Although the consensus chemical mechanism for FIH proposes that CTAD binding triggers O2-activation by causing the Fe(II) cofactor to release an aquo ligand, experimental evidence of this point has been absent. More broadly, this proposed coordination change at Fe(II) has not been observed during steady-state turnover in any αKG oxygenase to date. In this manuscript, solvent isotope effects (SIEs) were used as a direct mechanistic probe of substrate triggered aquo release in FIH, as inverse SIEs (SIE < 1) are signatures for pre-equilibrium aquo release from metal ions. Our mechanistic studies of FIH have revealed inverse solvent isotope effects in the steady-state rate constants at limiting concentrations of CTAD or αKG: D2Okcat/KM(CTAD) = 0.40 ± 0.07, D2Okcat/KM(αKG) = 0.32 ± 0.08, providing direct evidence for aquo release during steady-state turnover. Furthermore, the SIE at saturating concentrations of CTAD and αKG was inverse, D2Okcat = 0.51 ± 0.07, indicating that aquo release occurs after CTAD binds. The inverse kinetic SIEs observed in the steady state for FIH can be explained by a strong Fe-OH2 bond. The stable Fe-OH2 bond plays an important part in FIH’s regulatory role over O2 homeostasis in humans, and points toward a strategy for tightly coupling O2-activation with CTAD hydroxylation that relies on substrate triggering.
The factor inhibiting HIF (FIH) is one of the primary oxygen sensors in human cells, controlling gene expression by hydroxylating the α-subunit of the hypoxia inducible transcription factor (HIF). As FIH is an alpha-ketoglutarate dependent non-heme iron dioxygenase, oxygen activation is thought to precede substrate hydroxylation. The coupling between oxygen activation and substrate hydroxylation was hypothesized to be very tight, in order for FIH to fulfill its function as a regulatory enzyme. Coupling was investigated by looking for reactive oxygen species production during turnover. We used alkylsulfatase (AtsK), a metabolic bacterial enzyme with a related mechanism and similar turnover frequency, for comparison, and tested both FIH and AtsK for H2O2, O2− and OH• formation under steady and substrate-depleted conditions. Coupling ratios were determined by comparing the ratio of substrate consumed to product formed. We found that AtsK reacted with O2 on the seconds timescale in the absence of prime substrate, and uncoupled during turnover to produce H2O2; neither O2− nor OH• were detected. In contrast, FIH was unreactive toward O2 on the minutes timescale in the absence of prime substrate, and tightly coupled during steady-state turnover; we were unable to detect any reactive oxygen species produced by FIH. We also investigated the inactivation mechanisms of these enzymes and found that AtsK likely inactivated due to deoligomerizion, whereas FIH inactivated by slow autohydroxylation. Autohydroxylated FIH could not be reactivated by dithiothreitol (DTT) nor ascorbate, suggesting that autohydroxylation is likely to be irreversible under physiological conditions.
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