title: The second coordination sphere of FIH controls hydroxylation.
abstract: The factor inhibiting HIF (FIH) is a proximate oxygen sensor for human cells, hydroxylating Asn(803) 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 (His(2)Asp) facial triad, αKG, 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 αKG-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 αKG. 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+αKG)FIH indicated that these point mutations destabilized the π* orbitals of αKG, 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).
authors: Department of Chemistry, University of Massachusetts, Amherst, MA 01003, USA.
authors: Evren
authors: E
authors: Saban
authors: Yuan-Han
authors: YH
authors: Chen
authors: John A
authors: JA
authors: Hangasky
authors: Cornelius Y
authors: CY
authors: Taabazuing
authors: Breanne E
authors: BE
authors: Holmes
authors: Michael J
authors: MJ
authors: Knapp
mesh_terms: D000818:Animals; D002152:Calorimetry, Differential Scanning; D004578:Electron Spin Resonance Spectroscopy; D006900:Hydroxylation; D051379:Mice; D006899:Mixed Function Oxygenases / Q000737:chemistry / Q000502:physiology*; D008958:Models, Molecular; D017354:Point Mutation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013056:Spectrophotometry, Ultraviolet
chemical_list: D011994:Recombinant Proteins; D006899:Mixed Function Oxygenases; C551240:factor inhibiting hypoxia-inducible factor 1, mouse