The facial triad in the α-ketoglutarate dependent oxygenase FIH: A role for sterics in linking substrate binding to O2 activation.

The factor inhibiting hypoxia inducible factor-1α (FIH) is a nonheme Fe(II)/αKG oxygenase using a 2-His-1-Asp facial triad. FIH activates O 2 via oxidative decarboxylation of α-ketoglutarate (αKG) to generate an enzyme-based oxidant which hydroxylates the Asn 803 residue within the C-terminal transactivation domain (CTAD) of HIF-1α. Tight coupling of these two sequential reactions requires a structural linkage between the Fe(II) and the substrate binding site to ensure that O 2 activation occurs after substrate binds. We tested the hypothesis that the facial triad carboxylate (Asp 201 ) of FIH linked substrate binding and O 2 binding sites. Asp 201 variants of FIH were constructed and thoroughly characterized in vitro using steady-state kinetics, crystallography, autohydroxylation, and coupling measurements. Our studies revealed each variant activated O 2 with a catalytic efficiency similar to that of wild-type (WT) FIH ( k cat a K M(O 2 ) = 0.17 μM − 1 min − 1 ), but led to defects in the coupling of O 2 activation to substrate hydroxylation. Steady-state kinetics showed similar catalytic efficiencies for hydroxylation by WT-FIH ( k cat / K M(CTAD) = 0.42 μM − 1 min − 1 ) and D201G ( k cat / K M(CTAD) = 0.34 μM − 1 min − 1 ); hydroxylation by D201E was greatly impaired, while hydroxylation by D201A was undetectable. Analysis of the crystal structure of the D201E variant revealed steric crowding near the diffusible ligand site supporting a role for sterics from the facial triad carboxylate in the O 2 binding order. Our data support a model in which the facial triad carboxylate Asp 201 provides both steric and polar contacts to favor O 2 access to the Fe(II) only after substrate binds, leading to coupled turnover in FIH and other αKG oxygenases. [ABSTRACT FROM AUTHOR]