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Spectroscopic Studies of the Mononuclear Non-Heme FeII Enzyme FIH: Second-Sphere Contributions to Reactivity

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journal contribution
posted on 03.07.2013, 00:00 by Kenneth M. Light, John A. Hangasky, Michael J. Knapp, Edward I. Solomon
Factor inhibiting hypoxia-inducible factor (FIH) is an α-ketoglutarate (αKG)-dependent enzyme which catalyzes hydroxylation of residue Asn803 in the C-terminal transactivation domain (CAD) of hypoxia-inducible factor 1α (HIF-1α) and plays an important role in cellular oxygen sensing and hypoxic response. Circular dichroism (CD), magnetic circular dichroism (MCD), and variable-temperature, variable-field (VTVH) MCD spectroscopies are used to determine the geometric and electronic structures of FIH in its (FeII), (FeII/αKG), and (FeII/αKG/CAD) forms. (FeII)­FIH and (FeII/αKG)­FIH are found to be six-coordinate (6C), whereas (FeII/αKG/CAD)­FIH is found to be a 5C/6C mixture. Thus, FIH follows the general mechanistic strategy of non-heme FeII enzymes. Modeling shows that, when Arg238 of FIH is removed, the facial triad carboxylate binds to FeII in a bidentate mode with concomitant lengthening of the FeII/αKG carbonyl bond, which would inhibit the O2 reaction. Correlations over α-keto acid-dependent enzymes and with the extradiol dioxygenases show that members of these families (where both the electron source and O2 bind to FeII) have a second-sphere residue H-bonding to the terminal oxygen of the carboxylate, which stays monodentate. Alternatively, structures of the pterin-dependent and Rieske dioxygenases, which do not have substrate binding to FeII, lack H-bonds to the carboxylate and thus allow its bidentate coordination which would direct O2 reactivity. Finally, vis–UV MCD spectra show an unusually high-energy FeII → αKG π* metal-to-ligand charge transfer transition in (FeII/αKG)­FIH which is red-shifted upon CAD binding. This red shift indicates formation of H-bonds to the αKG that lower the energy of its carbonyl LUMO, activating it for nucleophilic attack by the Fe–O2 intermediate formed along the reaction coordinate.