Geometric and electronic structure contributions to function in non-heme iron enzymes

Abstract

Mononuclear non-heme Fe (NHFe) enzymes play key roles in DNA repair, the biosynthesis of antibiotics, the response to hypoxia, cancer therapy, and many other biological processes. These enzymes catalyze a diverse range of oxidation reactions, including hydroxylation, halogenation, ring closure, desaturation, and electrophilic aromatic substitution (EAS). Most of these enzymes use an Fe(II) site to activate dioxygen, but traditional spectroscopic methods have not allowed researchers to insightfully probe these ferrous active sites. We have developed a methodology that provides detailed geometric and electronic structure insights into these NHFe(II) active sites. Using these data, we have defined a general mechanistic strategy that many of these enzymes use: they control O2 activation (and limit autoxidation and self-hydroxylation) by allowing Fe(II) coordination unsaturation only in the presence of cosubstrates. Depending on the type of enzyme, O2 activation either involves a 2e(-) reduced Fe(III)-OOH intermediate or a 4e(-) reduced Fe(IV)═O intermediate. Nuclear resonance vibrational spectroscopy (NRVS) has provided the geometric structure of these intermediates, and magnetic circular dichroism (MCD) has defined the frontier molecular orbitals (FMOs), the electronic structure that controls reactivity. This Account emphasizes that experimental spectroscopy is critical in evaluating the results of electronic structure calculations. Therefore these data are a key mechanistic bridge between structure and reactivity. For the Fe(III)-OOH intermediates, the anticancer drug activated bleomycin (BLM) acts as the non-heme Fe analog of compound 0 in heme (e.g., P450) chemistry. However BLM shows different reactivity: the low-spin (LS) Fe(III)-OOH can directly abstract a H atom from DNA. The LS and high-spin (HS) Fe(III)-OOHs have fundamentally different transition states. The LS transition state goes through a hydroxyl radical, but the HS transition state is activated for EAS without O-O cleavage. This activation is important in one class of NHFe enzymes that utilizes a HS Fe(III)-OOH intermediate in dioxygenation. For Fe(IV)═O intermediates, the LS form has a π-type FMO activated for attack perpendicular to the Fe-O bond. However, the HS form (present in the NHFe enzymes) has a π FMO activated perpendicular to the Fe-O bond and a σ FMO positioned along the Fe-O bond. For the NHFe enzymes, the presence of π and σ FMOs enables enzymatic control in determining the type of reactivity: EAS or H-atom extraction for one substrate with different enzymes and halogenation or hydroxylation for one enzyme with different substrates.

FIGURE 1

NIR MCD transitions for NHFe^II sites. (A) Theoretical d orbital splittings for various geometries. (B) Experimentally-observed transitions.

FIGURE 2

VTVH MCD for Fe^II sites. (A) Splitting of the ⁵T_2g ground state upon axial and rhombic perturbations. S.O. Refers to the effect of spin-orbit coupling. (B) Nested MCD isotherms for an S = 2 non-Kramers doublet.

FIGURE 3

MCD studies of PAH. (A) MCD spectra of resting PAH (black) with substrate-bound (blue), pterin-bound (green) and substrate/pterin-bound (red). All spectra were collected at 5 K. (B) VTVH MCD data for resting (left) and substrate/pterin-bound (right) PAH. (C) Derived d-orbital energy level diagrams.

FIGURE 4

General mechanistic strategy for O₂-activation by NHFe^II enzymes.

FIGURE 5

(A) Steric and H-bonding contributions to loss of coordinated H₂O. CAD stands for C-terminal activation domain of the substrate HIF-1α. (B) Effect of a second-sphere H-bonding partner on the facial triad carboxylate.

FIGURE 6

Optimized structure of ABLM.

FIGURE 7

MCD spectral comparison of LMCT energies of (BLM)Fe^III (deprotonated amide to Fe^III) and a prototypical Fe^III-heme (porphyrin to Fe^III).

FIGURE 8

(A) NRVS spectra (PVDOS) of ¹⁶O and ¹⁸O (BLM)Fe^III (top) and DFT-simulated PVDOS spectra of two models with H₂O (middle) and OH⁻ (bottom) as axial ligands. (B) NRVS spectrum of ABLM (top) and DFT-simulated spectra with H₂O₂ (middle) and OOH⁻ (bottom) as axial ligands. (C) Major NRVS-active vibrational modes of ABLM.

FIGURE 9

(A) PES for H-atom abstraction by ABLM. (B) TS of direct H-atom abstraction. (C) FMOs of ABLM before (left) and at the TS (right).

FIGURE 10

(A) TSs of HS (TMC)Fe^III–OOH and LS (N4Py)Fe^III–OOH in H-atom abstraction. (B) PESs of HS (TMC)Fe^III–OOH and LS (N4Py)Fe^III–OOH O–O homolysis. Calculations were performed using the Polarizable Continuum Model (PCM) with a dielectric constant of acetone. (C) and (D) Forbidden and allowed orbital crossings for O–O bond homolysis of LS and HS Fe^III–alkylperoxo complexes. (See Ref for details)

FIGURE 11

MO diagram for S = 1 (TMC)Fe^IV=O (inset, top left) producing π* FMOs and σ* MO (right).

FIGURE 12

(A) 233 K Abs. and (B) VT MCD spectra of (TMC)Fe^IV=O. (C) Vibronic progression of band II (40 K; plotted positive).

FIGURE 13

MO diagrams of (TMC)Fe^IV=O in (A) S = 1 (ground) and (B) S = 2 states, showing that excitation of β-d_xy e⁻ into α-d_x²–y² orbital leads to spin-polarization of the α-manifold and a low-energy α-d_z² FMO for reactivity. (C) Isosurface plots of S = 2 FMOs.

FIGURE 14

233 K Abs. (top) and VT MCD (7T, bottom) spectra of (TMG₃tren)Fe^IV=O showing structured features corresponding to FMOs.

FIGURE 15

(left) PES of S = 2 states along the Fe—O coordinate for (TMG₃tren)Fe^IV=O, which lead to one σ and two π FMOs of Fe^III—oxyl character (right) at the TS.

FIGURE 16

Reactions of HPP with HPPD (left, EAS) and HmaS (right, H-atom abstraction) to produce homogentisate and (4-hydroxy)mandelate respectively.

FIGURE 17

CD spectra of HPP binding (red lines) to HmaS (top) and HPPD (bottom) Fe^II sites. Resting Fe^II d-d transitions (blue), and pyruvate-bound-Fe^II (green) as controls.

FIGURE 18

Reactivities of Fe^IV=O intermediates in HPPD (left) and HmaS (right), with different substrate (HPP) conformations leading to a σ TS for EAS in HPPD and a π TS for H-atom abstraction in HmaS. Formation of Fe^III—O^•− species at TS provides FMOs primed for reactivity.

FIGURE 19

SyrB2 catalyzes both chlorination of native substrate l-Thr (top) and hydroxylation of alternative substrate l-Nva (bottom).

FIGURE 20

Experimental (top) and simulated (middle) NRVS spectra of Fe^IV=O intermediate of SyrB2, ligated at Fe^IV=O active site by either Cl⁻ (green) or Br⁻ (red). Simulated spectra based on TBP structure with inert substrate l-Cpg (bottom).

FIGURE 21

O₂ activation in SyrB2 leading to two Fe^IV=O intermediate orientations relative to substrate (right): perpendicular (native l-Thr) and parallel (non-native l-Nva).

FIGURE 22

SyrB2 H-atom abstraction reaction coordinates for π-trajectory (green) and σ-trajectory (orange).