Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

In-crystal reaction cycle of a toluene-bound diiron hydroxylase

Abstract

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.

This is a preview of subscription content

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Figure 1: X-ray structures of T4moH and T4moHD.
Figure 2: X-ray structure of an oxygenated toluene intermediate in T4moHD.
Figure 3: Structure-correlated mechanism of diiron-enzyme aromatic ring hydroxylation.
Figure 4: A μ-1,1 ligand bound to Q228A T4moHD.

References

  1. 1

    Taylor, R. Electrophilic Aromatic Substitution (John Wiley & Sons, 1990)

  2. 2

    Roberts, K. M. & Fitzpatrick, P. F. Mechanisms of tryptophan and tyrosine hydroxylase. IUBMB Life 65, 350–357 (2013)

    CAS  Article  Google Scholar 

  3. 3

    Darbyshire, J. F., Iyer, K. R., Grogan, J., Korzekwa, K. R. & Trager, W. F. Substrate probe for the mechanism of aromatic hydroxylation catalyzed by cytochrome P450. Drug Metab. Dispos. 24, 1038–1045 (1996)

    CAS  PubMed  Google Scholar 

  4. 4

    Mitchell, K. H., Rogge, C. E., Gierahn, T. & Fox, B. G. Insight into the mechanism of aromatic hydroxylation by toluene 4-monooxygenase by use of specifically deuterated toluene and p-xylene. Proc. Natl Acad. Sci. USA 100, 3784–3789 (2003)

    CAS  ADS  Article  Google Scholar 

  5. 5

    Murray, L. J. et al. Characterization of the arene-oxidizing intermediate in ToMOH as a diiron(III) species. J. Am. Chem. Soc. 129, 14500–14510 (2007)

    CAS  Article  Google Scholar 

  6. 6

    Solomon, E. I., Light, K. M., Liu, L. V., Srnec, M. & Wong, S. D. Geometric and electronic structure contributions to function in non-heme iron enzymes. Acc. Chem. Res. 46, 2725–2739 (2013)

    CAS  Article  Google Scholar 

  7. 7

    Bathelt, C. M., Ridder, L., Mulholland, A. J. & Harvey, J. N. Aromatic hydroxylation by cytochrome P450: model calculations of mechanism and substituent effects. J. Am. Chem. Soc. 125, 15004–15005 (2003)

    CAS  Article  Google Scholar 

  8. 8

    Guroff, G. et al. Hydroxylation-induced migration: the NIH shift. Recent experiments reveal an unexpected and general result of enzymatic hydroxylation of aromatic compounds. Science 157, 1524–1530 (1967)

    CAS  ADS  Article  Google Scholar 

  9. 9

    Bailey, L. J., McCoy, J. G., Phillips, G. N., Jr. & Fox, B. G. Structural consequences of effector protein complex formation in a diiron hydroxylase. Proc. Natl Acad. Sci. USA 105, 19194–19198 (2008)

    CAS  ADS  Article  Google Scholar 

  10. 10

    Mitchell, K. H., Studts, J. M. & Fox, B. G. Combined participation of hydroxylase active site residues and effector protein binding in a para to ortho modulation of toluene 4-monooxygenase regiospecificity. Biochemistry 41, 3176–3188 (2002)

    CAS  Article  Google Scholar 

  11. 11

    Elsen, N. L., Moe, L. A., McMartin, L. A. & Fox, B. G. Redox and functional analysis of the Rieske ferredoxin component of the toluene 4-monooxygenase. Biochemistry 46, 976–986 (2007)

    CAS  Article  Google Scholar 

  12. 12

    Acheson, J. F., Bailey, L. J., Elsen, N. L. & Fox, B. G. Structural basis for biomolecular recognition in overlapping binding sites in a diiron enzyme system. Nat. Commun. 5, 5009 (2014)

    CAS  ADS  Article  Google Scholar 

  13. 13

    Bailey, L. J. et al. Crystallographic analysis of active site contributions to regiospecificity in the diiron enzyme toluene 4-monooxygenase. Biochemistry 51, 1101–1113 (2012)

    CAS  Article  Google Scholar 

  14. 14

    Bailey, L. J. & Fox, B. G. Crystallographic and catalytic studies of the peroxide-shunt reaction in a diiron hydroxylase. Biochemistry 48, 8932–8939 (2009)

    CAS  Article  Google Scholar 

  15. 15

    Liang, A. D., Wrobel, A. T. & Lippard, S. J. A flexible glutamine regulates the catalytic activity of toluene o-xylene monooxygenase. Biochemistry 53, 3585–3592 (2014)

    CAS  Article  Google Scholar 

  16. 16

    Elsen, N. L., Bailey, L. J., Hauser, A. D. & Fox, B. G. Role for threonine 201 in the catalytic cycle of the soluble diiron hydroxylase toluene 4-monooxygenase. Biochemistry 48, 3838–3846 (2009)

    CAS  Article  Google Scholar 

  17. 17

    Song, W. J. et al. Active site threonine facilitates proton transfer during dioxygen activation at the diiron center of toluene/o-xylene monooxygenase hydroxylase. J. Am. Chem. Soc. 132, 13582–13585 (2010)

    CAS  Article  Google Scholar 

  18. 18

    Gherman, B. F., Baik, M. H., Lippard, S. J. & Friesner, R. A. Dioxygen activation in methane monooxygenase: a theoretical study. J. Am. Chem. Soc. 126, 2978–2990 (2004)

    CAS  Article  Google Scholar 

  19. 19

    Solomon, E. I. & Park, K. Structure/function correlations over binuclear non-heme iron active sites. J. Biol. Inorg. Chem. 21, 575–588 (2016)

    CAS  Article  Google Scholar 

  20. 20

    Shan, X. & Que, L., Jr. Intermediates in the oxygenation of a nonheme diiron(II) complex, including the first evidence for a bound superoxo species. Proc. Natl Acad. Sci. USA 102, 5340–5345 (2005)

    CAS  ADS  Article  Google Scholar 

  21. 21

    Banerjee, R., Proshlyakov, Y., Lipscomb, J. D. & Proshlyakov, D. A. Structure of the key species in the enzymatic oxidation of methane to methanol. Nature 518, 431–434 (2015)

    CAS  ADS  Article  Google Scholar 

  22. 22

    Xing, G. et al. Evidence for C-H cleavage by an iron-superoxide complex in the glycol cleavage reaction catalyzed by myo-inositol oxygenase. Proc. Natl Acad. Sci. USA 103, 6130–6135 (2006)

    CAS  ADS  Article  Google Scholar 

  23. 23

    Wörsdörfer, B. et al. Organophosphonate-degrading PhnZ reveals an emerging family of HD domain mixed-valent diiron oxygenases. Proc. Natl Acad. Sci. USA 110, 18874–18879 (2013)

    ADS  Article  Google Scholar 

  24. 24

    Rivard, B. S. et al. Rate-determining attack on substrate precedes Rieske cluster oxidation during cis-dihydroxylation by benzoate dioxygenase. Biochemistry 54, 4652–4664 (2015)

    CAS  Article  Google Scholar 

  25. 25

    Capece, L., Lewis-Ballester, A., Yeh, S. R., Estrin, D. A. & Marti, M. A. Complete reaction mechanism of indoleamine 2,3-dioxygenase as revealed by QM/MM simulations. J. Phys. Chem. B 116, 1401–1413 (2012)

    CAS  Article  Google Scholar 

  26. 26

    Lovell, T., Li, J. & Noodleman, L. Density functional studies of oxidized and reduced methane monooxygenase. Optimized geometries and exchange coupling of active site clusters. Inorg. Chem. 40, 5251–5266 (2001)

    CAS  Article  Google Scholar 

  27. 27

    Lovell, T., Li, J. & Noodleman, L. Energetics of oxidized and reduced methane monooxygenase active site clusters in the protein environment. Inorg. Chem. 40, 5267–5278 (2001)

    CAS  Article  Google Scholar 

  28. 28

    Moche, M., Shanklin, J., Ghoshal, A. & Lindqvist, Y. Azide and acetate complexes plus two iron-depleted crystal structures of the di-iron enzyme Δ9 stearoyl-acyl carrier protein desaturase. Implications for oxygen activation and catalytic intermediates. J. Biol. Chem. 278, 25072–25080 (2003)

    CAS  Article  Google Scholar 

  29. 29

    Sazinsky, M. H., Bard, J., Di Donato, A. & Lippard, S. J. Crystal structure of the toluene/o-xylene monooxygenase hydroxylase from Pseudomonas stutzeri OX1. Insight into the substrate specificity, substrate channeling, and active site tuning of multicomponent monooxygenases. J. Biol. Chem. 279, 30600–30610 (2004)

    CAS  Article  Google Scholar 

  30. 30

    Vu, V. V. et al. Human deoxyhypusine hydroxylase, an enzyme involved in regulating cell growth, activates O2 with a nonheme diiron center. Proc. Natl Acad. Sci. USA 106, 14814–14819 (2009)

    CAS  ADS  Article  Google Scholar 

  31. 31

    Han, Z. et al. Crystal structure of the peroxo-diiron(III) intermediate of deoxyhypusine hydroxylase, an oxygenase involved in hypusination. Structure 23, 882–892 (2015)

    CAS  Article  Google Scholar 

  32. 32

    Broadwater, J. A., Ai, J., Loehr, T. M., Sanders-Loehr, J. & Fox, B. G. Peroxodiferric intermediate of stearoyl-acyl carrier protein Δ9 desaturase: oxidase reactivity during single turnover and implications for the mechanism of desaturation. Biochemistry 37, 14664–14671 (1998)

    CAS  Article  Google Scholar 

  33. 33

    Knoot, C. J., Kovaleva, E. G. & Lipscomb, J. D. Crystal structure of CmlI, the arylamine oxygenase from the chloramphenicol biosynthetic pathway. J. Biol. Inorg. Chem. 21, 589–603 (2016)

    CAS  Article  Google Scholar 

  34. 34

    Yun, D. et al. (Mu-1,2-peroxo)diiron(III/III) complex as a precursor to the diiron(III/IV) intermediate X in the assembly of the iron-radical cofactor of ribonucleotide reductase from mouse. Biochemistry 46, 1925–1932 (2007)

    CAS  Article  Google Scholar 

  35. 35

    Moënne-Loccoz, P. et al. The ferroxidase reaction of ferritin reveals a diferric μ-1,2 bridging peroxide intermediate in common with other O2-activating non-heme diiron proteins. Biochemistry 38, 5290–5295 (1999)

    Article  Google Scholar 

  36. 36

    Moe, L. A. et al. Remarkable aliphatic hydroxylation by the diiron enzyme toluene 4-monooxygenase in reactions with radical or cation diagnostic probes norcarane, 1,1-dimethylcyclopropane, and 1,1-diethylcyclopropane. Biochemistry 43, 15688–15701 (2004)

    CAS  Article  Google Scholar 

  37. 37

    Otwinowski, Z. & Minor, W. The processing of X-ray diffraction data collected in oscillation mode. Methods Enzymol. 276, 307–326 (1997)

    CAS  Article  Google Scholar 

  38. 38

    Vagin, A. & Teplyakov, A. Molecular replacement with MOLREP. Acta Crystallogr. D Biol. Crystallogr. 66, 22–25 (2010)

    CAS  Article  Google Scholar 

  39. 39

    Adams, P. D. et al. Advances, interactions, and future developments in the CNS, Phenix, and Rosetta structural biology software systems. Annu. Rev. Biophys. 42, 265–287 (2013)

    CAS  Article  Google Scholar 

  40. 40

    Emsley, P. & Cowtan, K. Coot: model-building tools for molecular graphics. Acta Crystallogr. D Biol. Crystallogr. 60, 2126–2132 (2004)

    Article  Google Scholar 

  41. 41

    Chen, V. B. et al. MolProbity: all-atom structure validation for macromolecular crystallography. Acta Crystallogr. D Biol. Crystallogr. 66, 12–21 (2010)

    CAS  Article  Google Scholar 

  42. 42

    DeLano, W. L. The PyMOL Molecular Graphics System (DeLano Scientific, 2002)

  43. 43

    ORCA. An Ab initio, Density Functional, and Semiempirical Program Package (Version 3.0.3; Max-Planck-Institute for Chemical Energy Conversion, Germany, 2014)

  44. 44

    Becke, A. D. Density-functional exchange-energy approximation with correct asymptotic behavior. Phys. Rev. A. Gen. Phys. 38, 3098–3100 (1988)

    CAS  ADS  Article  Google Scholar 

  45. 45

    Lee, C., Yang, W. & Parr, R. G. Development of the Colle-Salvetti correlation-energy formula into a functional of the electron density. Phys. Rev. B. Condens. Matter. 37, 785–789 (1988)

    CAS  ADS  Article  Google Scholar 

  46. 46

    Schäfer, A., Horn, H. & Ahlrichs, R. Fully optimized contracted Gaussian-basis sets for atoms Li to Kr. J. Chem. Phys. 97, 2571–2577 (1992)

    ADS  Article  Google Scholar 

  47. 47

    Schäfer, A., Huber, C. & Ahlrichs, R. Fully optimized contracted Gaussian basis sets of triple zeta valence quality for atoms Li to Kr. J. Chem. Phys. 100, 5829–5835 (1994)

    ADS  Article  Google Scholar 

  48. 48

    Sievers, F. et al. Fast, scalable generation of high-quality protein multiple sequence alignments using Clustal Omega. Mol. Syst. Biol. 7, 539 (2011)

    Article  Google Scholar 

  49. 49

    Robert, X. & Gouet, P. Deciphering key features in protein structures with the new ENDscript server. Nucleic Acids Res. 42, W320–324 (2014)

    CAS  Article  Google Scholar 

Download references

Acknowledgements

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.

Author information

Affiliations

Authors

Contributions

J.F.A. and L.J.B. designed biochemical experiments, prepared enzyme samples, obtained crystals, solved and refined structures, analysed data, and wrote the manuscript. T.C.B. performed DFT calculations, analysed data, and wrote the manuscript. B.G.F. led the project, designed biochemical experiments, analysed data, and wrote the manuscript. All authors discussed results and commented on the manuscript.

Corresponding author

Correspondence to Brian G. Fox.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Additional information

Reviewer Information Nature thanks S. de Visser, J. Lipscomb and L. Que for their contribution to the peer review of this work.

Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data figures and tables

Extended Data Figure 1 Alternative images of the T4moH–toluene complex.

The T4moH active site is shown with bound toluene defined by a FoFc omit map (purple, 3σ). Active site residues, waters and cofactors are shown with a 2FoFc 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.

Extended Data Figure 3 Peroxo intermediates formed in T4moHD.

The diiron centre, two glutamate ligands and waters are shown. Bond distances (Å) are indicated. a, μ-η22 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.

Extended Data Figure 4 Alternative images of the T4moHD oxygenated toluene intermediate.

A 2FoFc map is shown for all active site residues, cofactors, waters, and ligands (light blue, contour 1.0σ). FoFc omit maps are shown for ligands (purple, contour 3.0σ). a, b, Superposition of the active site 2FoFc and toluene and peroxo FoFc omit maps. c, Superposition of the active site 2FoFc and toluene FoFc omit maps. d. Superposition of the active site 2FoFc and peroxo FoFc 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.

FoFc omit maps of the μ-1,1 O–O intermediate are shown at the different contour levels indicated. A 2FoFc map is shown for active site residues, cofactors and waters (light blue, contour 1.0σ).

Extended Data Table 1 Data collection and refinement statistics
Extended Data Table 2 Kinetic analysis of T4moH reactions

PowerPoint slides

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

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

Download citation

Further reading

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing