The Second Coordination Sphere of FIH Controls Hydroxylation

Abstract

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.

Inverse Solvent Isotope Effects Arising from Substrate Triggering in the Factor Inhibiting Hypoxia Inducible Factor

“…In close agreement with previously reported values,(14, 31) the K M(αKG) remained constant at 20 ± 2μM for all CTAD concentrations. The regression plot of k cat /K M(αKG) as a function of [CTAD] passed through the origin, as expected for ordered sequential binding of αKG prior to CTAD (Figure 3).…”

“…Previously we showed tight coupling between succinate production and CTAD hydroxylation in H 2 O. (14) Here we observed that FIH’s two half reactions remained tightly coupled in D 2 O, with C equal to unity within experimental uncertainty( C = 1.1 ± 0.1) (Table 2). …”

Biological Outer-Sphere Coordination

“…The sites are primed for reactivity with O 2 by the binding of a-KG, which serves as a bidentate ligand to Fe. A recent study [16] examined the role of OSC in the reactivity of one member of this family, "factor inhibiting HIF-1" (FIH). FIH participates in human O 2 sensing by catalyzing hydroxylation of C b of Asn803 in the C-terminal transactivation domain (CTAD) of hypoxia-inducible factor (HIF) [17].…”

Substrate Promotes Productive Gas Binding in the α-Ketoglutarate-Dependent Oxygenase FIH

“…In light of the fact that primary substrate does not bind directly to Fe 2+ , and the observation that contacts throughout the active site greatly impact reactivity, 15,55 it appears that substrate binding alters the steric and non-covalent contacts near the gas binding site to stimulate O 2 activation. Understanding factors governing O 2 binding are key to understanding why the ferryl forms preferentially after the primary substrate is in position for subsequent hydroxylation.…”

“…10,11 A key feature of FIH function is that O 2 activation is stimulated by the binding of the CTAD domain of the HIF transcription factor, 12 which ensures that the rate of CTAD hydroxylation is directly proportional to the concentration of O 2 (Scheme 1). 13–15 Recently, it was shown that CTAD binding leads to a mixture of 6-coordinate (6C) and 5-coordinate (5C) Fe 2+ in FIH/CTAD, 7 suggesting that greater O 2 access to the Fe 2+ may only partially explain the increased O 2 reactivity upon CTAD binding. The rate-limiting step for O 2 activation by FIH occurs before early in turnover, making FIH an excellent enzyme to interrogate the link between O 2 activation and substrate hydroxylation in the broad class of αKG oxygenases.…”