Protein Flexibility of the α-Ketoglutarate-Dependent Oxygenase Factor-Inhibiting HIF-1: Implications for Substrate Binding, Catalysis, and Regulation.

Protein dynamics are crucial for the mechanistically ordered enzymes to bind to their substrate in the correct sequence and perform catalysis. Factor-inhibiting HIF-1 (FIH) is a nonheme Fe(II) α-ketoglutarate-dependent oxygenase that is a key hypoxia (low pO2) sensor in humans. As these hypoxia-sensing enzymes follow a multistep chemical mechanism consuming α-ketoglutarate, a protein substrate that is hydroxylated, and O2, understanding protein flexibility and the order of substrate binding may aid in the development of strategies for selective targeting. The primary substrate of FIH is the C-terminal transactivation domain (CTAD) of hypoxia-inducible factor 1α (HIF) that is hydroxylated on the side chain of Asn803. We assessed changes in protein flexibility connected to metal and αKG binding, finding that (M+αKG) binding significantly stabilized the cupin barrel core of FIH as evidenced by enhanced thermal stability and decreased protein dynamics as assessed by global amide hydrogen/deuterium exchange mass spectrometry and limited proteolysis. Confirming predictions of the consensus mechanism, (M+αKG) increased the affinity of FIH for CTAD as measured by titrations monitoring intrinsic tryptophan fluorescence. The decreased protein dynamics caused by (M+αKG) enforces a sequentially ordered substrate binding sequence in which αKG binds before CTAD, suggesting that selective inhibition may require inhibitors that target the binding sites of both αKG and the prime substrate. A consequence of the correlation between dynamics and αKG binding is that all relevant ligands must be included in binding-based inhibitor screens, as shown by testing permutations of M, αKG, and inhibitor.

Chloride Supports O2 Activation in the D201G Facial Triad Variant of Factor-Inhibiting Hypoxia Inducible Factor, an α-Ketoglutarate Dependent Oxygenase.

α-Ketoglutarate (αKG) dependent oxygenases comprise a large superfamily of enzymes that activate O2 for varied reactions. While most of these enzymes contain a nonheme Fe bound by a His2(Asp/Glu) facial triad, a small number of αKG-dependent halogenases require only the two His ligands to bind Fe and activate O2. The enzyme "factor inhibiting HIF" (FIH) contains a His2Asp facial triad and selectively hydroxylates polypeptides; however, removal of the Asp ligand in the Asp201→Gly variant leads to a highly active enzyme, seemingly without a complete facial triad. Herein, we report on the formation of an Fe-Cl cofactor structure for the Asp201→Gly FIH variant using X-ray absorption spectroscopy (XAS), which provides insight into the structure of the His2Cl facial triad found in halogenases. The Asp201→Gly variant supports anion dependent peptide hydroxylation, demonstrating the requirement for a complete His2X facial triad to support O2 reactivity. Our results indicated that exogenous ligand binding to form a complete His2X facial triad was essential for O2 activation and provides a structural model for the His2Cl-bound nonheme Fe found in halogenases.

O2 Activation by Nonheme FeII α-Ketoglutarate-Dependent Enzyme Variants: Elucidating the Role of the Facial Triad Carboxylate in FIH.

FIH [factor inhibiting HIF (hypoxia inducible factor)] is an α-ketoglutarate (αKG)-dependent nonheme iron enzyme that catalyzes the hydroxylation of the C-terminal transactivation domain (CAD) asparagine residue in HIF-1α to regulate cellular oxygen levels. The role of the facial triad carboxylate ligand in O2 activation and catalysis was evaluated by replacing the Asp201 residue with Gly (D201G), Ala (D201A), and Glu (D201E). Magnetic circular dichroism (MCD) spectroscopy showed that the (FeII)FIH variants were all 6-coordinate (6C) and the αKG plus CAD bound FIH variants were all 5-coordinate (5C), mirroring the behavior of the wild-type ( wt) enzyme. When only αKG is bound, all FIH variants exhibited weaker FeII-OH2 bonds for the sixth ligand compared to wt, and for αKG-bound D201E this is either extremely weak or the site is 5C, demonstrating that the Asp201 residue plays an important role in the wt enzyme in ensuring that the (FeII/αKG)FIH site remains 6C. Variable-temperature, variable-field (VTVH) MCD spectroscopy showed that all of the αKG- and CAD-bound FIH variants, though 5C, have different ground-state geometric and electronic structures, which impair their oxygen activation rates. Comparison of O2 consumption to substrate hydroxylation kinetics revealed uncoupling between the two half reactions in the variants. Thus, the Asp201 residue also ensures fidelity between CAD substrate binding and oxygen activation, enabling tightly coupled turnover.

Investigations on the role of a solvent tunnel in the α-ketoglutarate dependent oxygenase factor inhibiting HIF (FIH).

Non-heme Fe(II)/α-ketoglutarate (αKG)-dependent oxygenases catalyze a wide array of reactions through coupling oxidative decarboxylation of αKG to substrate oxygenation. This class of enzymes follows a sequential mechanism in which O2 reacts only after binding primary substrate, raising questions over how protein structure tailors molecular access to the Fe(II) cofactor. The enzyme "factor inhibiting hypoxia inducible factor" (FIH) senses pO2 in human cells by hydroxylating the C-terminal transactivation domain (CTAD), suggesting that structural elements limiting molecular access to the active site may limit the pO2 response. In this study, we tested the impact of a solvent-accessible tunnel in FIH on molecular access to the active site in FIH. The size of the tunnel was increased through alanine point mutagenesis (Y93A, E105A, and Q147A), followed by a suite of mechanistic and spectroscopic probes. Steady-state kinetics varying O2 or CTAD indicated that O2 passage through the tunnel was not affected by Ala substitutions, allowing us to conclude that this narrow tunnel did not impact pO2 sensing by FIH. Steady-state kinetics with varied αKG concentrations revealed increased substrate inhibition for the Ala variants, suggesting that a second αKG molecule may bind near the active site of FIH. If this solvent-accessible tunnel is the O2 entry tunnel, it may be narrow in order to permit O2 access while preventing metabolic intermediates, such as αKG, from inhibiting FIH under physiological conditions.