The Second Coordination Sphere of FIH Controls Hydroxylation

The factor inhibiting HIF (FIH) is a proximate oxygen sensor for human cells, hydroxylating Asn(803) within the alpha-subunit of the hypoxia inducible factor (HIF). FIH is an alpha-ketoglutatrate (alpha KG)-dependent, non-heme Fe(11) dioxygenase, in which Fe(II) is coordinated by a (His(2)Asp) facial triad, alpha KG, and H(2)O. Hydrogen bonding among the facial triad, the HIF-Asn(803) side chain, 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 alpha KG-centered (Asn(205) and Asn(294)) or HIF-Asn(803)-centered (Arg(238) and Gln(239)) second-sphere residues. The second sphere was tested for local effects on priming Fe(II) to react with O(2), 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 alpha KG. Asn(205)-> Ala and Asn(294)-> Ala mutants exhibited diminished rates of steady-state turnover, while minimally affecting autohydroxylation, consistent with impaired oxidative decarboxylation. Blue-shifted metal to ligand charge transfer transitions for (Fe+alpha KG)FIH indicated that these point mutations destabilized the pi* orbitals of alpha KG, further supporting a slowed rate of oxidative decarboxylation. The Arg(238)-> Met mutant exhibited steady-state rates too low to measure and diminished product yields, suggesting impaired substrate positioning or priming; the Arg(238)-> Met mutant was capable of O(2) activation for the autohydroxylation reaction. The Gln(239)-> Asn mutant exhibited significantly slowed steady-state kinetics and diminished product yields, suggesting impaired substrate positioning or priming. As HIF binding to the Gln(239)-> Asn mutant stimulated autohydroxylation, it is more likely that this point mutant simply mispositions the HIF-Asn(803) side chain. This work combines kinetics and spectroscopy to show that these second-sphere hydrogen bonds play roles in promoting oxidative decarboxylation, priming Fe(II) to bind O(2), and positioning HIF-Asn(803).