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

Factor inhibiting hypoxia-inducible factor (FIH) is an alpha-ketoglutarate (alpha KG)-dependent enzyme which catalyzes hydroxylation of residue Asn803 in the C-terminal transactivation domain (CAD) of hypoxia-inducible factor 1 alpha (HIF-1 alpha) 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 (Fe-II), (Fe-II/alpha KG), and (Fe-II/alpha KG/CAD) forms. (Fe-II)FIH and (Fe-II/alpha KG)FIH are found to be six-coordinate (6C), whereas (Fe-II/alpha KG/CAD)FIH is found to be a 5C/6C mixture. Thus, FIH follows the general mechanistic strategy of non-heme Fe-II enzymes. Modeling shows that, when Arg238 of FIH is removed, the facial triad carboxylate binds to Fe-II in a bidentate mode with concomitant lengthening of the Fe-II/alpha KG carbonyl bond, which would inhibit the O-2 reaction. Correlations over a-keto acid-dependent enzymes and with the extradiol dioxygenases show that members of these families (where both the electron source and O-2 bind to Fe-II) 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 Fe-II, lack H-bonds to the carboxylate and thus allow its bidentate coordination which would direct O-2 reactivity. Finally, vis-UV MCD spectra show an unusually high-energy Fe-II -> alpha KG pi* metal-to-ligand charge transfer transition in (Fe-II/alpha KG)FIH which is red-shifted upon CAD binding. This red shift indicates formation of H-bonds to the alpha KG that lower the energy of its carbonyl LUMO, activating it for nucleophilic attack by the Fe-O-2 intermediate formed along the reaction coordinate.