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.

  • Letter
  • Published:

Elucidation of the Fe(iv)=O intermediate in the catalytic cycle of the halogenase SyrB2

Abstract

Mononuclear non-haem iron (NHFe) enzymes catalyse a broad range of oxidative reactions, including halogenation, hydroxylation, ring closure, desaturation and aromatic ring cleavage reactions. They are involved in a number of biological processes, including phenylalanine metabolism, the production of neurotransmitters, the hypoxic response and the biosynthesis of secondary metabolites1,2,3. The reactive intermediate in the catalytic cycles of these enzymes is a high-spin S = 2 Fe(iv)=O species, which has been trapped for a number of NHFe enzymes4,5,6,7,8, including the halogenase SyrB2 (syringomycin biosynthesis enzyme 2). Computational studies aimed at understanding the reactivity of this Fe(iv)=O intermediate9,10,11,12,13 are limited in applicability owing to the paucity of experimental knowledge about its geometric and electronic structure. Synchrotron-based nuclear resonance vibrational spectroscopy (NRVS) is a sensitive and effective method that defines the dependence of the vibrational modes involving Fe on the nature of the Fe(iv)=O active site14,15,16. Here we present NRVS structural characterization of the reactive Fe(iv)=O intermediate of a NHFe enzyme, namely the halogenase SyrB2 from the bacterium Pseudomonas syringae pv. syringae. This intermediate reacts via an initial hydrogen-atom abstraction step, performing subsequent halogenation of the native substrate or hydroxylation of non-native substrates17. A correlation of the experimental NRVS data to electronic structure calculations indicates that the substrate directs the orientation of the Fe(iv)=O intermediate, presenting specific frontier molecular orbitals that can activate either selective halogenation or hydroxylation.

This is a preview of subscription content, access via your institution

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Catalytic cycle of αKG-dependent NHFe enzymes.
Figure 2: NRVS PVDOS spectra of SyrB2–Cl and SyrB2–Br.
Figure 3: Computational spectra and structure of five-coordinate TBP structural candidate 1Cpg–X for the Fe(iv)=O intermediate of SyrB2.
Figure 4: DFT-predicted normal modes of
Figure 5: Hydrogen-atom abstraction reaction coordinates.

Similar content being viewed by others

References

  1. Solomon, E. I. et al. Geometric and electronic structure/function correlations in non-heme iron enzymes. Chem. Rev. 100, 235–350 (2000)

    Article  CAS  PubMed  Google Scholar 

  2. Costas, M., Mehn, M. P., Jensen, M. P. & Que, L., Jr Dioxygen activation at mononuclear nonheme iron active sites: enzymes, models, and intermediates. Chem. Rev. 104, 939–986 (2004)

    Article  CAS  PubMed  Google Scholar 

  3. Vaillancourt, F. H., Yeh, E., Vosburg, D. A., Garneau-Tsodikova, S. & Walsh, C. T. Nature’s inventory of halogenation catalysts: oxidative strategies predominate. Chem. Rev. 106, 3364–3378 (2006)

    Article  CAS  PubMed  Google Scholar 

  4. Krebs, C., Galonić Fujimori, D., Walsh, C. T. & Bollinger, J. M., Jr Non-heme Fe(IV)-oxo intermediates. Acc. Chem. Res. 40, 484–492 (2007)

    Article  CAS  PubMed  Google Scholar 

  5. Eser, B. E. et al. Direct spectroscopic evidence for a high-spin Fe(IV) intermediate in tyrosine hydroxylase. J. Am. Chem. Soc. 129, 11334–11335 (2007)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Galonić Fujimori, D. et al. Spectroscopic evidence for a high-spin Br-Fe(IV)-oxo intermediate in the alpha-ketoglutarate-dependent halogenase CytC3 from Streptomyces. J. Am. Chem. Soc. 129, 13408–13409 (2007)

    Article  CAS  Google Scholar 

  7. Matthews, M. L. et al. Substrate-triggered formation and remarkable stability of the C−H bond-cleaving chloroferryl intermediate in the aliphatic halogenase, SyrB2. Biochemistry 48, 4331–4343 (2009)

    Article  CAS  PubMed  Google Scholar 

  8. Panay, A. J., Lee, M., Krebs, C., Bollinger, J. M., Jr & Fitzpatrick, P. F. Evidence for a high-spin Fe(IV) species in the catalytic cycle of a bacterial phenylalanine hydroxylase. Biochemistry 50, 1928–1933 (2011)

    Article  CAS  PubMed  Google Scholar 

  9. Pandian, S., Vincent, M. A., Hillier, I. H. & Burton, N. A. Why does the enzyme SyrB2 chlorinate, but does not hydroxylate, saturated hydrocarbons? A density functional theory (DFT) study. Dalton Trans. 6201–6207 (2009)

  10. Kulik, H. J., Blasiak, L. C., Marzari, N. & Drennan, C. L. First-principles study of non-heme Fe(II) halogenase SyrB2 reactivity. J. Am. Chem. Soc. 131, 14426–14433 (2009)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. de Visser, S. P. & Latifi, R. Carbon dioxide: a waste product in the catalytic cycle of alpha-ketoglutarate dependent halogenases prevents the formation of hydroxylated by-products. J. Phys. Chem. B 113, 12–14 (2009)

    Article  CAS  PubMed  Google Scholar 

  12. Borowski, T., Noack, H., Radoń, M., Zych, K. & Siegbahn, P. E. M. Mechanism of selective halogenation by SyrB2: a computational study. J. Am. Chem. Soc. 132, 12887–12898 (2010)

    Article  CAS  PubMed  Google Scholar 

  13. Usharani, D., Janardanan, D. & Shaik, S. Does the TauD enzyme always hydroxylate alkanes, while an analogous synthetic non-heme reagent always desaturates them? J. Am. Chem. Soc. 133, 176–179 (2011)

    Article  CAS  PubMed  Google Scholar 

  14. Bell, C. B. et al. A combined NRVS and DFT study of FeIV=O model complexes: a diagnostic method for the elucidation of non-heme iron enzyme intermediates. Angew. Chem. Int. Edn 47, 9071–9074 (2008)

    Article  CAS  Google Scholar 

  15. Wong, S. D. et al. Nuclear resonance vibrational spectroscopy on the FeIV=O S = 2 non-heme site in TMG3tren: experimentally calibrated insights into reactivity. Angew. Chem. Int. Edn 50, 3215–3218 (2011)

    Article  CAS  Google Scholar 

  16. Park, K. et al. Nuclear resonance vibrational spectroscopic and computational study of high-valent diiron complexes relevant to enzyme intermediates. Proc. Natl Acad. Sci. USA 110, 6275–6280 (2013)

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  17. Matthews, M. L. et al. Substrate positioning controls the partition between halogenation and hydroxylation in the aliphatic halogenase, SyrB2. Proc. Natl Acad. Sci. USA 106, 17723–17728 (2009)

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  18. Vaillancourt, F. H., Yin, J. & Walsh, C. T. SyrB2 in syringomycin E biosynthesis is a nonheme FeII α-ketoglutarate- and O2-dependent halogenase. Proc. Natl Acad. Sci. USA 102, 10111–10116 (2005)

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  19. Blasiak, L. C., Vaillancourt, F. H., Walsh, C. T. & Drennan, C. L. Crystal structure of the non-haem iron halogenase SyrB2 in syringomycin biosynthesis. Nature 440, 368–371 (2006)

    Article  ADS  CAS  PubMed  Google Scholar 

  20. Krebs, C. et al. Novel approaches for the accumulation of oxygenated intermediates to multi-millimolar concentrations. Coord. Chem. Rev. 257, 234–243 (2013)

    Article  CAS  Google Scholar 

  21. Seto, M., Yoda, Y., Kikuta, S., Zhang, X. & Ando, M. Observation of nuclear resonant scattering accompanied by phonon excitation using synchrotron radiation. Phys. Rev. Lett. 74, 3828–3831 (1995)

    Article  ADS  CAS  PubMed  Google Scholar 

  22. Chumakov, A. I. & Sturhahn, W. Experimental aspects of inelastic nuclear resonance scattering. Hyperfine Interact. 123/124, 781–808 (1999)

    Article  Google Scholar 

  23. Sage, J. T. et al. Nuclear resonance vibrational spectroscopy of a protein active-site mimic. J. Phys. Condens. Matter 13, 7707–7722 (2001)

    Article  ADS  CAS  Google Scholar 

  24. Diebold, A. R. et al. Activation of α-keto acid-dependent dioxygenases: application of an {FeNO}7/{FeO2}8 methodology for characterizing the initial steps of O2 activation. J. Am. Chem. Soc. 133, 18148–18160 (2011)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Neidig, M. L. et al. Spectroscopic and electronic structure studies of aromatic electrophilic attack and hydrogen-atom abstraction by non-heme iron enzymes. Proc. Natl Acad. Sci. USA 103, 12966–12973 (2006)

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  26. Srnec, M., Wong, S. D., England, J., Que, L. & Solomon, E. I. π-frontier molecular orbitals in S = 2 ferryl species and elucidation of their contributions to reactivity. Proc. Natl Acad. Sci. USA 109, 14326–14331 (2012)

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  27. Comba, P. & Wunderlich, S. Iron-catalyzed halogenation of alkanes: modeling of nonheme halogenases by experiment and DFT calculations. Chemistry 16, 7293–7299 (2010)

    Article  CAS  PubMed  Google Scholar 

  28. Alp, E. E., Mooney, T. M., Toellner, T. & Sturhahn, W. Nuclear resonant scattering beamline at the Advanced Photon Source. Hyperfine Interact. 90, 323–334 (1994)

    Article  ADS  Google Scholar 

  29. Yoda, Y. et al. Nuclear resonant scattering beamline at SPring-8. Nucl. Instrum. Methods A 467–468, 715–718 (2001)

    Article  ADS  Google Scholar 

  30. Ahlrichs, R., Bär, M., Häser, M., Horn, H. & Kölmel, C. Electronic structure calculations on workstation computers: the program system Turbomole. Chem. Phys. Lett. 162, 165–169 (1989)

    Article  ADS  CAS  Google Scholar 

  31. Frisch, M. J. et al. Gaussian 09, Revision A.1 (Gaussian, Wallingford, 2009)

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

    Article  ADS  CAS  Google Scholar 

  33. Perdew, J. Density-functional approximation for the correlation energy of the inhomogeneous electron gas. Phys. Rev. B 33, 8822–8824 (1986)

    Article  ADS  CAS  Google Scholar 

  34. Vosko, S. H., Wilk, L. & Nusair, M. Accurate spin-dependent electron liquid correlation energies for local spin density calculations: a critical analysis. Can. J. Phys. 58, 1200–1211 (1980)

    Article  ADS  CAS  Google Scholar 

  35. Weigend, F. & Ahlrichs, R. Balanced basis sets of split valence, triple zeta valence and quadruple zeta valence quality for H to Rn: design and assessment of accuracy. Phys. Chem. Chem. Phys. 7, 3297–3305 (2005)

    Article  CAS  PubMed  Google Scholar 

  36. Eichkorn, K., Treutler, O., Öhm, H., Häser, M. & Ahlrichs, R. Auxiliary basis sets to approximate Coulomb potentials. Chem. Phys. Lett. 240, 283–290 (1995)

    Article  ADS  CAS  Google Scholar 

  37. Eichkorn, K., Weigend, F., Treutler, O. & Ahlrichs, R. Auxiliary basis sets for main row atoms and transition metals and their use to approximate Coulomb potentials. Theor. Chim. Acta 97, 119–124 (1997)

    Article  CAS  Google Scholar 

  38. Klamt, A. & Schüürmann, G. COSMO: a new approach to dielectric screening in solvents with explicit expressions for the screening energy and its gradient. J. Chem. Soc. Perkin Trans. 2 0, 799–805 (1993)

    Article  CAS  Google Scholar 

  39. Schäfer, A., Klamt, A., Sattel, D., Lohrenz, J. C. W. & Eckert, F. COSMO implementation in Turbomole: extension of an efficient quantum chemical code towards liquid systems. Phys. Chem. Chem. Phys. 2, 2187–2193 (2000)

    Article  Google Scholar 

  40. Wachters, A. Gaussian basis set for molecular wavefunctions containing third-row atoms. J. Chem. Phys. 52, 1033–1036 (1970)

    Article  ADS  CAS  Google Scholar 

  41. Hay, P. J. Gaussian basis sets for molecular calculations. The representation of 3d orbitals in transition-metal atoms. J. Chem. Phys. 66, 4377–4384 (1977)

    Article  ADS  CAS  Google Scholar 

  42. McLean, A. D. & Chandler, G. S. Contracted Gaussian basis sets for molecular calculations. I. Second row atoms, Z = 11–18. J. Chem. Phys. 72, 5639–5648 (1980)

    Article  ADS  CAS  Google Scholar 

  43. Krishnan, R., Binkley, J. S., Seeger, R. & Pople, J. A. Self-consistent molecular orbital methods. XX. A basis set for correlated wave functions. J. Chem. Phys. 72, 650–654 (1980)

    Article  ADS  CAS  Google Scholar 

  44. Mennucci, B. & Tomasi, J. Continuum solvation models: a new approach to the problem of solute’s charge distribution and cavity boundaries. J. Chem. Phys. 106, 5151–5158 (1997)

    Article  ADS  CAS  Google Scholar 

  45. Mennucci, B., Cancès, E. & Tomasi, J. Evaluation of solvent effects in isotropic and anisotropic dielectrics and in ionic solutions with a unified integral equation method: theoretical bases, computational implementation, and numerical applications. J. Phys. Chem. B 101, 10506–10517 (1997)

    Article  CAS  Google Scholar 

  46. Cammi, R., Mennucci, B. & Tomasi, J. Second-order Møller−Plesset analytical derivatives for the polarizable continuum model using the relaxed density approach. J. Phys. Chem. A 103, 9100–9108 (1999)

    Article  CAS  Google Scholar 

  47. Cammi, R., Mennucci, B. & Tomasi, J. Fast evaluation of geometries and properties of excited molecules in solution: a Tamm-Dancoff model with application to 4-dimethylamino-enzonitrile. J. Phys. Chem. A 104, 5631–5637 (2000)

    Article  CAS  Google Scholar 

  48. Leu, B. M. et al. Quantitative vibrational dynamics of iron in nitrosyl porphyrins. J. Am. Chem. Soc. 126, 4211–4227 (2004)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Tenderholt, A. gennrvs (2009); Pymol script available at http://www.stanford.edu/group/solomon/gennrvs/gennrvs.py.txt

  50. Srnec, M. et al. Structural and spectroscopic properties of the peroxodiferric intermediate of Ricinus communis soluble Δ9 desaturase. Inorg. Chem. 51, 2806–2820 (2012)

    Article  CAS  PubMed  Google Scholar 

  51. Becke, A. D. Density-functional thermochemistry. III. The role of exact exchange. J. Chem. Phys. 98, 5648–5652 (1993)

    Article  ADS  CAS  Google Scholar 

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

    Article  ADS  CAS  Google Scholar 

  53. Miehlich, B., Savin, A., Stoll, H. & Preuss, H. Results obtained with the correlation energy density functionals of Becke and Lee, Yang and Parr. Chem. Phys. Lett. 157, 200–206 (1989)

    Article  ADS  CAS  Google Scholar 

  54. Grimme, S. Accurate description of van der Waals complexes by density functional theory including empirical corrections. J. Comput. Chem. 25, 1463–1473 (2004)

    Article  CAS  PubMed  Google Scholar 

  55. Grimme, S. Semiempirical GGA-type density functional constructed with a long-range dispersion correction. J. Comput. Chem. 27, 1787–1799 (2006)

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

Funding for this work was provided by the National Institutes of Health (GM-40392 to E.I.S. and GM-69657 to J.M.B. and C.K.) and the National Science Foundation (MCB-0919027 to E.I.S., and MCB-642058 and CHE-724084 to J.M.B. and C.K.). Work at the Advanced Photon Source was supported by the Department of Energy, Office of Science, under contract DE-AC-02-06CH11357. Synchrotron experiments at SPring-8 were performed with the approval of the Japan Synchrotron Radiation Research Institute (JASRI; proposal no. 2010B1569). M.S. thanks the Rulíšek group at the IOCB, Prague, for use of their computational resources.

Author information

Authors and Affiliations

Authors

Contributions

S.D.W. and M. Srnec contributed equally to this work. E.I.S., C.K. and J.M.B. designed the experiments. S.D.W., M. Srnec, M.L.M., L.V.L., Y.K., K.P. and C.B.B. performed the experiments. S.D.W., M. Srnec and E.I.S. analysed the data and wrote the manuscript. E.E.A., J.Z., Y.Y., S.K. and M. Seto provided technical assistance at the sychrotron beamlines.

Corresponding authors

Correspondence to Carsten Krebs, J. Martin Bollinger or Edward I. Solomon.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Information

This file contains Supplementary Figures 1-11 and Supplementary Table 1. (PDF 7114 kb)

PowerPoint slides

Rights and permissions

Reprints and permissions

About this article

Cite this article

Wong, S., Srnec, M., Matthews, M. et al. Elucidation of the Fe(iv)=O intermediate in the catalytic cycle of the halogenase SyrB2. Nature 499, 320–323 (2013). https://doi.org/10.1038/nature12304

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nature12304

This article is cited by

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