Controlled Oxygen Activation in Human Oxygen Sensor FIH

One of the primary oxygen sensors in human cells, which controls gene expression by hydroxylating the hypoxia inducible transcription factor (HIFα) is the factor inhibiting HIF (FIH). 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. Alkylsulfatase (AtsK), a metabolic bacterial enzyme with a related mechanism and similar turnover frequency, was used for comparison, and both FIH and AtsK were tested 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. 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; any reactive oxygen species produced by FIH was not available for detection. Inactivation mechanisms of these enzymes were also investigated. AtsK likely inactivated due to deoligomerization, whereas FIH inactivated by slow autohydroxylation. Autohydroxylated FIH could not be reactivated by dithiothreitol (DTT) nor is ascorbate, suggesting that autohydroxylation likely to be irreversible under physiological conditions. Iron in the FIH active site 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-state 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. Asn205àAla and Asn294àAla 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 Arg238àMet mutant exhibited steady-state rates too low to measure and diminished product yields, suggesting impaired substrate positioning or priming; Arg238àMet was capable of O2-activation for the autohydroxylation reaction. The Gln239àAsn mutant exhibited significantly slowed steady-state kinetics and diminished product yields, suggesting impaired substrate positioning or priming. As HIF binding to Gln239àAsn stimulated autohydroxylation, it is more likely that this point mutant simply mis-positions the HIF-Asn803 sidechain. By combining kinetics and spectroscopy, it was shown that these second sphere hydrogen bonds play roles in promoting oxidative decarboxylation, priming Fe(II) to bind O2, and positioning HIF-Asn803