Substrate Positioning by Gln(239) Stimulates Turnover in Factor Inhibiting HIF, an αKG-Dependent Hydroxylase

[Image: see text] Nonheme Fe(II)/αKG-dependent oxygenases catalyze diverse reactions, typically inserting an O atom from O(2) into a C–H bond. Although the key to their catalytic cycle is the fact that binding and positioning of primary substrate precede O(2) activation, the means by which substrate binding stimulates turnover is not well understood. Factor Inhibiting HIF (FIH) is a Fe(II)/αKG-dependent oxygenase that acts as a cellular oxygen sensor in humans by hydroxylating the target residue Asn(803), found in the C-terminal transactivation domain (CTAD) of hypoxia inducible factor-1. FIH-Gln(239) makes two hydrogen bonds with CTAD-Asn(803), positioning this target residue over the Fe(II). We hypothesized the positioning of the side chain of CTAD-Asn(803) by FIH-Gln(239) was critical for stimulating O(2) activation and subsequent substrate hydroxylation. The steady-state characterization of five FIH-Gln(239) variants (Ala, Asn, Glu, His, and Leu) tested the role of hydrogen bonding potential and sterics near the target residue. Each variant exhibited a 20–1200-fold decrease in k(cat) and k(cat)/K(M(CTAD)), but no change in K(M(CTAD)), indicating that the step after CTAD binding was affected by point mutation. Uncoupled O(2) activation was prominent in these variants, as shown by large coupling ratios (C = [succinate]/[CTAD-OH] = 3–5) for each of the FIH-Gln(239) → X variants. The coupling ratios decreased in D(2)O, indicating an isotope-sensitive inactivation for variants, not observed in the wild type. The data presented indicate that the proper positioning of CTAD-Asn(803) by FIH-Gln(239) is necessary to suppress uncoupled turnover and to support substrate hydroxylation, suggesting substrate positioning may be crucial for directing O(2) reactivity within the broader class of αKG hydroxylases.