New Frontiers in Nonheme Enzymatic Oxyferryl Species

Abstract

Non-heme mononuclear iron dependent (NHM-Fe) enzymes exhibit exceedingly diverse catalytic reactivities. Despite their catalytic versatilities, the mononuclear iron centers in these enzymes show a relatively simple architecture, in which an iron atom is ligated with 2-4 amino acid residues, including histidine, aspartic or glutamic acid. In the past two decades, a common high-valent reactive iron intermediate, the S=2 oxyferryl (Fe(IV)-oxo or Fe(IV)=O) species, has been repeatedly discovered in NHM-Fe enzymes containing a 2-His-Fe or 2-His-1-carboxylate-Fe center. However, for 3-His/4-His-Fe enzymes, no common reactive intermediate has been identified. Recently, we have spectroscopically characterized the first S=1 Fe(IV) intermediate in a 3-His-Fe containing enzyme, OvoA, which catalyzes a novel oxidative carbon-sulfur bond formation. In this review, we summarize the broad reactivities demonstrated by S=2 Fe(IV)-oxo intermediates, the discovery of the first S=1 Fe(IV) intermediate in OvoA and the mechanistic implication of such a discovery, and the intrinsic reactivity differences of the S=2 and the S=1 Fe(IV)-oxo species. Finally, we postulate the possible reasons to utilize an S=1 Fe(IV) species in OvoA and their implications to other 3-His/4-His-Fe enzymes.

Keywords: C−H activation; Ferryl intermediates; Nonheme iron enzymes; Oxidative C−S bond formation; Spin state.

Figure 1.

Iron center structures of TauD in the resting Fe(II) state and of the oxyferryl intermediate established by spectroscopic and protein crystallographic studies. (a) top: iron center structure with 2OG and taurine binding derived from protein crystallographic studies (ref); bottom: optical absorption features of Fe(II)-2OG MLCT band in TauD (ref.). (b) clockwise: the structural model of the oxyferryl intermediate (TauD J) in TauD based on spectroscopic and computational studies (ref.^,); the Mössbauer spectrum (ref.), the resonance Raman data (ref.), and the EXAFS data of J (ref.). The data are reproduced with permissions from ref 12, , , and . Copyright 1999, 2003, and 2004 American Chemical Society.

Figure 2.

Schematic illustration of the pathways of HAT by an oxyferryl intermediate. Left: the orbital overlap model for the Fe(IV)=O moiety. Middle: the electron distribution on the frontier molecular orbitals for both S = 1 and S = 2 spin states based on a 6-coordinate Fe(IV) center in a pseudo-octahedral geometry. The electron transfer route in the σ-, or the π-pathway from the activated C-H bond to the Fe(IV) center is also shown. The stability of the spin state (S = 1 or S = 2) is governed by the energetics of the dσ_x2-y2* orbital, which is modulated by the equatorial ligand field strength. Right: the C-H bond orientation relative to the Fe(IV)=O moiety in both the σ-, and the π-pathway.

Figure 3.

Left: iron center structure of SyrB2 (PDB: 2FCT); Right: mechanisms of hydroxylation vs. halogenation via σ- or π-pathway. The inline and the offline configuration of the chloro-oxyferryl intermediate are shown. In the inline configuration, the substrate C-H bond is positioned parallel to the Fe(IV)=O moiety (σ configuration), while in the offline configuration, the C-H bond is positioned perpendicular to the Fe(IV)=O moiety (π configuration).

Figure 4.

Left: the iron center structure of OvoA from Hydrogenimonas thermophila in the presence of substrates Cys and His (PDB:8KHQ). Note that in this published crystal structure contains cobalt instead of iron at the metal center. Right: the reactions catalyzed by OvoA and EgtB.

Figure 5.

Spectroscopic evidence supporting Int490 as an S = 1 Fe(IV) species. (a) Stopped-flow absorption spectra of the OvoA_Mtht•Fe(II)•Cys•His complex reacting with an O₂ saturated buffer. Inset: Single wavelength A490 time traces of the OvoA_Mtht reaction with Cys and His (black), with Cys and d5-His (blue), and with Cys and His in the presence of Cld reacting with an O₂ buffer containing chlorite (red). (b) 4.2 K zero field ⁵⁷Fe Mössbauer spectra of the OvoA reaction freeze-quenched at various time points an O₂ saturated buffer. (c) Variable field and variable temperature ⁵⁷Fe Mössbauer spectra of the sample freeze quenched at 0.02 s (O₂ was generated by the Cld/chlorite system). The top spectrum is the spectrum of the sample measured at zero applied field while the bottom three spectra are the different spectra showing only the spectral contribution of Int490. In b and c, black = experimentally obtained data, grey = total spectral simulations, red = Int490 sub-spectra, blue = EP complex sub-spectra. (d) The normalized iron K-edge XANES spectra of the OvoA_Mtht•Fe(II)•Cys•His complex (black) and Int490 (red) after removing the contribution of the Fe(II) quaternary complex.

Figure 6.

(a) The structural model of Int490 based on DFT calculations; (b) reaction mechanisms of OvoA-catalyzed reaction.

Scheme 1.

Consensus mechanism of Fe(II)-2OG enzyme catalyzed hydroxylation

Scheme 2.

Reaction mechanism of Fe(II)-2OG enzyme catalyzed desaturation.

Scheme 3.

(a) The overall reaction catalyzed by IPNS. (b) Consensus reaction mechanism of IPNS-catalyzed reaction. AA = amino acid.

Scheme 4.

Selected examples of synthetic oxyferryl complexes in an S = 1 spin state (left panel), and in an S = 2 spin in either a pseudo-octahedral geometry (right top panel) or a trigonal bipyramidal geometry (right bottom panel). The electron distributions on the frontier molecular orbitals for each case are shown.