The Second Coordination Sphere of FIH Controls Hydroxylation

• Published in: Biochemistry (Easton) Vol. 50; no. 21; pp. 4733 - 4740
• Main Authors: Saban, Evren, Chen, Yuan-Han, A. Hangasky, John, Y. Taabazuing, Cornelius, Holmes, Breanne E, Knapp, Michael J
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 31.05.2011
• Subjects: Animals; Calorimetry, Differential Scanning; Electron Spin Resonance Spectroscopy; Hydroxylation; Mice; Mixed Function Oxygenases - chemistry; Mixed Function Oxygenases - physiology; Models, Molecular; Point Mutation; Recombinant Proteins - chemistry; Recombinant Proteins - metabolism; Spectrophotometry, Ultraviolet
• ISSN: 0006-2960; 1520-4995; 1520-4995
• DOI: 10.1021/bi102042t

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 among the facial triad, the HIF-Asn803 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 (Asn205 and Asn294) or HIF-Asn803-centered (Arg238 and 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 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 Arg238 → Met mutant exhibited steady-state rates too low to measure and diminished product yields, suggesting impaired substrate positioning or priming; the Arg238 → Met mutant 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 the Gln239 → Asn mutant stimulated autohydroxylation, it is more likely that this point mutant simply mispositions the HIF-Asn803 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 O2, and positioning HIF-Asn803.