Electrophilic aromatic substitution is one of the most important and recognizable classes of organic chemical transformation. Enzymes create the strong electrophiles that are needed for these highly energetic reactions by using O2, electrons, and metals or other cofactors. Although the nature of the oxidants that carry out electrophilic aromatic substitution has been deduced from many approaches, it has been difficult to determine their structures. Here we show the structure of a diiron hydroxylase intermediate formed during a reaction with toluene. Density functional theory geometry optimizations of an active site model reveal that the intermediate is an arylperoxo Fe2+/Fe3+ species with delocalized aryl radical character. The structure suggests that a carboxylate ligand of the diiron centre may trigger homolytic cleavage of the O–O bond by transferring a proton from a metal-bound water. Our work provides the spatial and electronic constraints needed to propose a comprehensive mechanism for diiron enzyme arene hydroxylation that accounts for many prior experimental results.
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This work was funded by the National Science Foundation MCB-0843239 (B.G.F.). Use of the Advanced Photon Source was supported by the US Department of Energy, Office of Science Contract No. W-31-109-ENG-38. Use of the Life Science Collaborative Access Team (LS-CAT) was supported by the College of Agricultural and Life Sciences, Department of Biochemistry, and Graduate School of the University of Wisconsin. J.F.A. received a Wisconsin Distinguished Graduate Fellowship in support of this work.
The authors declare no competing financial interests.
Reviewer Information Nature thanks S. de Visser, J. Lipscomb and L. Que for their contribution to the peer review of this work.
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Extended data figures and tables
The T4moH active site is shown with bound toluene defined by a Fo−Fc omit map (purple, 3σ). Active site residues, waters and cofactors are shown with a 2Fo−Fc map (light blue, 1.5σ). Mobile diiron ligands E104 and E231 are highlighted (white sticks). HOH1 binds in a putative O2 binding site.
Extended Data Figure 2 Sequence alignment of the α-subunits of diiron hydroxylases with determined structures.
The residues coordinating the diiron centre are marked with black stars. Other active site residues are marked with black diamonds. All aromatic ring hydroxylases contain a glutamine at position 228, whereas methane monooxygenase has a glutamate at this position. The figure was prepared with ClustalOmega48 and ESPript 349.
The diiron centre, two glutamate ligands and waters are shown. Bond distances (Å) are indicated. a, μ-η2:η2 arylperoxo Fe2+/Fe3+ intermediate with radical character in the aromatic ring formed from the reaction of reduced T4moHD with O2 in the presence of toluene. b, cis-μ-1,2 peroxo diferric intermediate formed from the reaction of diferric T4moHD with excess H2O2, from PDB 3I6314. c, μ-1,1 (hydro)peroxo diferric intermediate formed from the reaction of reduced Gln228Ala T4moHD with O2 in the absence of toluene.
A 2Fo−Fc map is shown for all active site residues, cofactors, waters, and ligands (light blue, contour 1.0σ). Fo−Fc omit maps are shown for ligands (purple, contour 3.0σ). a, b, Superposition of the active site 2Fo−Fc and toluene and peroxo Fo−Fc omit maps. c, Superposition of the active site 2Fo−Fc and toluene Fo−Fc omit maps. d. Superposition of the active site 2Fo−Fc and peroxo Fo−Fc omit maps.
Extended Data Figure 5 An alternative image of the DFT-optimized model of the T4moHD oxygenated substrate intermediate.
The computed spin density distribution (blue for positive and red for negative) and Löwdin spin populations for relevant atoms are indicated. Green asterisks mark the atoms that were kept fixed (along with the two Fe atoms) during the partial geometry optimization.
Extended Data Figure 6 Images showing different views of the DFT-optimized model for the enzyme–product complex produced after O–O bond homolysis but before rearomatization of the aromatic ring.
The computed spin density distribution (blue for positive and red for negative) and Löwdin spin populations for relevant atoms are indicated.
Extended Data Figure 7 Alternative images of the μ-1,1 hydroperoxo intermediate formed in Q228A T4moHD.
Fo−Fc omit maps of the μ-1,1 O–O intermediate are shown at the different contour levels indicated. A 2Fo−Fc map is shown for active site residues, cofactors and waters (light blue, contour 1.0σ).
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Acheson, J., Bailey, L., Brunold, T. et al. In-crystal reaction cycle of a toluene-bound diiron hydroxylase. Nature 544, 191–195 (2017). https://doi.org/10.1038/nature21681
Scientific Reports (2017)