title: Spectroscopic studies of the mononuclear non-heme Fe(II) enzyme FIH: second-sphere contributions to reactivity.
abstract: 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 (Fe(II)), (Fe(II)/αKG), and (Fe(II)/αKG/CAD) forms. (Fe(II))FIH and (Fe(II)/αKG)FIH are found to be six-coordinate (6C), whereas (Fe(II)/αKG/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)/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 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 O2 reactivity. Finally, vis-UV MCD spectra show an unusually high-energy Fe(II) → αKG π* metal-to-ligand charge transfer transition in (Fe(II)/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.
authors.affiliation: Department of Chemistry, Stanford University, Stanford, California 94305, USA.
authors.forename: Kenneth M
authors.initials: KM
authors.lastname: Light
authors.forename: John A
authors.initials: JA
authors.lastname: Hangasky
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Knapp
authors.forename: Edward I
authors.initials: EI
authors.lastname: Solomon
mesh_terms: D002942:Circular Dichroism; D018360:Crystallography, X-Ray; D006801:Humans; D006899:Mixed Function Oxygenases / Q000737:chemistry* / Q000378:metabolism; D008958:Models, Molecular; D011789:Quantum Theory; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012097:Repressor Proteins / Q000737:chemistry* / Q000378:metabolism; D019265:Spectroscopy, Near-Infrared
chemical_list: D011994:Recombinant Proteins; D012097:Repressor Proteins; D006899:Mixed Function Oxygenases; C439790:HIF1AN protein, human