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 (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 nonheme 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 shows 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.

Mono- and binuclear non-heme iron chemistry from a theoretical perspective

“…[16] TDDFT was also found to be helpful for understanding the observed changes in Fe/α-ketoglutarate (α-KG) π* metal-to-ligand charge transfer upon variations of H-bonding, and 5/6-fold coordination, which reflect stabilization of the α-KG π* orbital directly influencing O2-reactivity in α-KG-dependent enzymes. [17] Figure 2. Structures of some ligands employed in biomimetic non-heme iron complexes.…”

Substrate Promotes Productive Gas Binding in the α-Ketoglutarate-Dependent Oxygenase FIH

“…Simulations of the EPR spectra were calculated using the XSophe software package version 1.1.14. 33 As the S = 3/2 {FeNO} 7 spin is in the weak field limit (D≫hν), D was fixed at 10 cm −1 , corresponding to the measured and calculated values of D for {FeNO} 7 adducts of related enzymes 4-hydroxyphenylpyruvate dioxygenase (HPPD) (~ 8 cm −1 ) and TauD (~ 12 cm −1 ). 16,17 …”

“…34 Methyl imidazoles were used for His 199 and His 279 , propionic acid for Asp 201 , 2 oxopropionate for αKG, and Asn 803 was truncated and contained a methyl group from the adjacent amino acid (Figure 6). The {FeNO} 7 S = 3/2 spin state was generated using the spin-coupling scheme of a high spin Fe 3+ (S Fe = 5/2) antiferromagnetically coupled to two electrons from the NO − π* orbitals (S NO = 1) as this represents the proper electronic description of ferrous NO complexes. 16,20 Using the geometry optimized structures, a rigid potential energy surface scan (B3LYP/6-311+g(2d,p)) was performed to investigate the energy barrier for rotating the bent NO ligand in the axial coordination position of FIH.…”

“…10,11 A key feature of FIH function is that O 2 activation is stimulated by the binding of the CTAD domain of the HIF transcription factor, 12 which ensures that the rate of CTAD hydroxylation is directly proportional to the concentration of O 2 (Scheme 1). 13–15 Recently, it was shown that CTAD binding leads to a mixture of 6-coordinate (6C) and 5-coordinate (5C) Fe 2+ in FIH/CTAD, 7 suggesting that greater O 2 access to the Fe 2+ may only partially explain the increased O 2 reactivity upon CTAD binding. The rate-limiting step for O 2 activation by FIH occurs before early in turnover, making FIH an excellent enzyme to interrogate the link between O 2 activation and substrate hydroxylation in the broad class of αKG oxygenases.…”

“…The structure of FIH was solved with the O 2 analogue NO bound to Fe, offering the first direct insight into the gas binding geometry in this enzyme. Through a combination of DFT calculations, {FeNO} 7 EPR spectroscopy, and UV-Vis absorption spectroscopy we demonstrate that CTAD binding stimulates O 2 reactivity by altering the orientation of the bound gas molecule. Although FIH binds NO with moderate affinity, the bound gas can adopt either of two orientations with similar stability; upon CTAD binding, NO adopts a single preferred orientation that is appropriate to support oxidative decarboxylation.…”