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.

RETRACTED ARTICLE: Endoperoxide formation by an α-ketoglutarate-dependent mononuclear non-haem iron enzyme

This article was retracted on 19 May 2021

This article has been updated

Abstract

Many peroxy-containing secondary metabolites1,2 have been isolated and shown to provide beneficial effects to human health3,4,5. Yet, the mechanisms of most endoperoxide biosyntheses are not well understood. Although endoperoxides have been suggested as key reaction intermediates in several cases6,7,8, the only well-characterized endoperoxide biosynthetic enzyme is prostaglandin H synthase, a haem-containing enzyme9. Fumitremorgin B endoperoxidase (FtmOx1) from Aspergillus fumigatus is the first reported α-ketoglutarate-dependent mononuclear non-haem iron enzyme that can catalyse an endoperoxide formation reaction10,11,12. To elucidate the mechanistic details for this unique chemical transformation, we report the X-ray crystal structures of FtmOx1 and the binary complexes it forms with either the co-substrate (α-ketoglutarate) or the substrate (fumitremorgin B). Uniquely, after α-ketoglutarate has bound to the mononuclear iron centre in a bidentate fashion, the remaining open site for oxygen binding and activation is shielded from the substrate or the solvent by a tyrosine residue (Y224). Upon replacing Y224 with alanine or phenylalanine, the FtmOx1 catalysis diverts from endoperoxide formation to the more commonly observed hydroxylation. Subsequent characterizations by a combination of stopped-flow optical absorption spectroscopy and freeze-quench electron paramagnetic resonance spectroscopy support the presence of transient radical species in FtmOx1 catalysis. Our results help to unravel the novel mechanism for this endoperoxide formation reaction.

Your institute does not have access to this article

Relevant articles

Open Access articles citing this article.

Access options

Buy article

Get time limited or full article access on ReadCube.

$32.00

All prices are NET prices.

Figure 1: Enzymatic characterization of wild-type FtmOx1.
Figure 2: Structures of FtmOx1.
Figure 3: Characterization of Y224A- and Y224F-substituted FtmOx1.
Figure 4: Proposed FtmOx1 mechanistic model.
Figure 5: Evidence for transient radical species in the reaction pathway.

Accession codes

Primary accessions

Protein Data Bank

Data deposits

The structural factors and coordinates of FtmOx1 and its complexes with either α-KG or fumitremorgin B have been deposited in the Protein Data Bank with accession codes 4Y5T, 4Y5S and 4ZON.

Change history

References

  1. Casteel, D. A. Peroxy natural products. Nat. Prod. Rep. 9, 289–312 (1992)

    CAS  PubMed  Article  Google Scholar 

  2. Casteel, D. A. Peroxy natural products. Nat. Prod. Rep. 16, 55–73 (1999)

    Article  Google Scholar 

  3. Chaturvedi, D., Goswami, A., Saikia, P. P., Barua, N. C. & Rao, P. G. Artemisinin and its derivatives: a novel class of anti-malarial and anti-cancer agents. Chem. Soc. Rev. 39, 435–454 (2010)

    CAS  PubMed  Article  Google Scholar 

  4. Paddon, C. J. & Keasling, J. D. Semi-synthetic artemisinin: a model for the use of synthetic biology in pharmaceutical development. Nature Rev. Microbiol. 12, 355–367 (2014)

    CAS  Article  Google Scholar 

  5. Dembitsky, V. M. Bioactive peroxides as potential therapeutic agents. Eur. J. Med. Chem. 43, 223–251 (2008)

    CAS  PubMed  Article  Google Scholar 

  6. Widboom, P. F., Fielding, E. N., Liu, Y. & Bruner, S. D. Structural basis for cofactor-independent dioxygenation in vancomycin biosynthesis. Nature 447, 342–345 (2007)

    ADS  CAS  PubMed  Article  Google Scholar 

  7. Steiner, R. A., Janssen, H. J., Roversi, P., Oakley, A. J. & Fetzner, S. Structural basis for cofactor-independent dioxygenation of N-heteroaromatic compounds at the α/β-hydrolase fold. Proc. Natl Acad. Sci. USA 107, 657–662 (2010)

    ADS  CAS  PubMed  Article  Google Scholar 

  8. Thierbach, S. et al. Substrate-assisted O2 activation in a cofactor-independent dioxygenase. Chem. Biol. 21, 217–225 (2014)

    CAS  PubMed  Article  Google Scholar 

  9. Marnett, L. J. Cyclooxygenase mechanisms. Curr. Opin. Chem. Biol. 4, 545–552 (2000)

    CAS  PubMed  Article  Google Scholar 

  10. Grundmann, A. & Li, S. M. Overproduction, purification and characterization of FtmPT1, a brevianamide F prenyltransferase from Aspergillus fumigatus . Microbiology 151, 2199–2207 (2005)

    CAS  PubMed  Article  Google Scholar 

  11. Steffan, N., Grundmann, A., Afiyatullov, S., Ruan, H. & Li, S. M. FtmOx1, a non-heme Fe(II) and α-ketoglutarate-dependent dioxygenase, catalyses the endoperoxide formation of verruculogen in Aspergillus fumigatus . Org. Biomol. Chem. 7, 4082–4087 (2009)

    CAS  PubMed  Article  Google Scholar 

  12. Kato, N. et al. Gene disruption and biochemical characterization of verruculogen synthase of Aspergillus fumigatus . ChemBioChem 12, 711–714 (2011)

    CAS  PubMed  Article  Google Scholar 

  13. Clifton, I.J. et al. Structural studies on 2-oxoglutarate oxygenases and related double-stranded α-helix fold proteins. J. Inorg. Biochem. 100, 644–669 (2006)

    CAS  PubMed  Article  Google Scholar 

  14. Hausinger, R. P. Fe(II)/α-ketoglutarate-dependent hydroxylases and related enzymes. Crit. Rev. Biochem. Mol. Biol. 39, 21–68 (2004)

    CAS  PubMed  Article  Google Scholar 

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

    CAS  PubMed  Article  Google Scholar 

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

    CAS  PubMed  Article  Google Scholar 

  17. Kovaleva, E. G. & Lipscomb, J. D. Versatility of biological non-heme Fe(II) centers in oxygen activation reactions. Nature Chem. Biol. 4, 186–193 (2008)

    CAS  Google Scholar 

  18. Ryle, M. J., Padmakumar, R. & Hausinger, R. P. Stopped-flow kinetic analysis of Escherichia coli taurine/α-ketoglutarate dioxygenase: interactions with α-ketoglutarate, taurine, and oxygen. Biochemistry 38, 15278–15286 (1999)

    CAS  PubMed  Article  Google Scholar 

  19. Liu, A., Ho, R. Y. N. & Que, L. Alternative reactivity of an α-ketoglutarate-dependent Iron(II) oxygenase: enzyme self-hydroxylation. J. Am. Chem. Soc. 123, 5126–5127 (2001)

    CAS  PubMed  Article  Google Scholar 

  20. Volkamer, A., Kuhn, D., Grombacher, T., Rippmann, F. & Rarey, M. Combining global and local measures for structure-based druggability predictions. J. Chem. Inf. Model. 52, 360–372 (2012)

    CAS  PubMed  Article  Google Scholar 

  21. Elkins, J. M. et al. X-ray crystal structure of Escherichia coli taurine/α-ketoglutarate dioxygenase complexed to ferrous iron and substrates. Biochemistry 41, 5185–5192 (2002)

    CAS  PubMed  Article  Google Scholar 

  22. Liu, P. et al. Protein purification and function assignment of the epoxidase catalyzing the formation of fosfomycin. J. Am. Chem. Soc. 123, 4619–4620 (2001)

    CAS  PubMed  Article  Google Scholar 

  23. Vaillancourt, F. H., Yeh, E., Vosburg, D. A., O’Connor, S. E. & Walsh, C. T. Cryptic chlorination by a non-haem iron enzyme during cyclopropyl amino acid biosynthesis. Nature 436, 1191–1194 (2005)

    ADS  CAS  PubMed  Article  Google Scholar 

  24. 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)

    ADS  CAS  PubMed  Article  Google Scholar 

  25. Clifton, I. J. et al. Crystal structure of carbapenem synthase (CarC). J. Biol. Chem. 278, 20843–20850 (2003)

    CAS  PubMed  Article  Google Scholar 

  26. Chang, W. C. et al. Mechanism of the C5 stereoinversion reaction in the biosynthesis of carbapenem antibiotics. Science 343, 1140–1144 (2014)

    ADS  CAS  PubMed  PubMed Central  Article  Google Scholar 

  27. Blodgett, J. A. V. et al. Unusual transformations in the biosynthesis of the antibiotic phosphinothricin tripeptide. Nature Chem. Biol. 3, 480–485 (2007)

    CAS  Article  Google Scholar 

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

    CAS  PubMed  Article  Google Scholar 

  29. Stubbe, J. & van der Donk, W. A. Protein radicals in enzyme catalysis. Chem. Rev. 98, 705–762 (1998)

    CAS  PubMed  Article  Google Scholar 

  30. Minor, W. & Otwinowski, Z. in Methods in Enzymology, Macromolecular Crystallography (Academic Press, 1997)

  31. Adams, P. D. et al. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr. D 66, 213–221 (2010)

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  32. Terwilliger, T. C. Maximum-likelihood density modification. Acta Crystallogr. D 56, 965–972 (2000)

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  33. Terwilliger, T. C. et al. Decision-making in structure solution using Bayesian estimates of map quality: the PHENIX AutoSol wizard. Acta Crystallogr. D 65, 582–601 (2009)

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  34. Baker, D., Bystroff, C., Fletterick, R. J. & Agard, D. A. PRISM: topologically constrained phased refinement for macromolecular crystallography. Acta Crystallogr. D 49, 429–439 (1993)

    CAS  PubMed  Article  Google Scholar 

  35. Bricogne, G. Geometric sources of redundancy in intensity data and their use for phase determination. Acta Crystallogr. A 30, 395–405 (1974)

    ADS  CAS  Article  Google Scholar 

  36. Brünger, A. T. Free R value: a novel statistical quantity for assessing the accuracy of crystal structures. Nature 355, 472–475 (1992)

    ADS  PubMed  Article  Google Scholar 

  37. Cowtan, K. Error estimation and bias correction in phase-improvement calculations. Acta Crystallogr. D 55, 1555–1567 (1999)

    CAS  PubMed  Article  Google Scholar 

  38. Cowtan, K. D. & Main, P. Improvement of macromolecular electron-density maps by the simultaneous application of real and reciprocal space constraints. Acta Crystallogr. D 49, 148–157 (1993)

    CAS  PubMed  Article  Google Scholar 

  39. Cowtan, K. D. & Main, P. Phase combination and cross validation in iterated density-modification calculations. Acta Crystallogr. D 52, 43–48 (1996)

    CAS  PubMed  Article  Google Scholar 

  40. Winn, M. D. et al. Overview of the CCP4 suite and current developments. Acta Crystallogr. D 67, 235–242 (2011)

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  41. Sayre, D. Least-squares phase refinement. II. High-resolution phasing of a small protein. Acta Crystallogr. A 30, 180–184 (1974)

    ADS  Article  Google Scholar 

  42. Schuller, D. J. MAGICSQUASH: more versatile non-crystallographic averaging with mulitple constraints. Acta Crystallogr. D 52, 425–434 (1996)

    CAS  PubMed  Article  Google Scholar 

  43. Swanson, S. M. Core tracing: depicting connections between features in electron density. Acta Crystallogr. D 50, 695–708 (1994)

    CAS  PubMed  Article  Google Scholar 

  44. Wang, B. C. Resolution of phase ambiguity in macromolecular crystallography. Methods Enzymol. 115, 90–112 (1985)

    CAS  PubMed  Article  Google Scholar 

  45. Zhang, K. & Main, P. The use of Sayre’s equation with solvent flattening and histogram matching for phase extension and refinement of protein structures. Acta Crystallogr. A 46, 377–381 (1990)

    Article  Google Scholar 

  46. Cowtan, K. The Buccaneer software for automated model building. 1. Tracing protein chains. Acta Crystallogr. D 62, 1002–1011 (2006)

    PubMed  Article  CAS  Google Scholar 

  47. Cowtan, K. Fitting molecular fragments into electron density. Acta Crystallogr. D 64, 83–89 (2008)

    CAS  PubMed  Article  Google Scholar 

  48. Emsley, P., Lohkamp, B., Scott, W. G. & Cowtan, K. Features and development of Coot. Acta Crystallogr. D 66, 486–501 (2010)

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  49. Afonine, P. V. et al. Towards automated crystallographic structure refinement with phenix.refine. Acta Crystallogr. D 68, 352–367 (2012)

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  50. Berkholz, D. S., Shapovalov, M. V., Dunbrack, R. L., Jr & Karplus, P. A. Conformation dependence of backbone geometry in proteins. Structure 17, 1316–1325 (2009)

    CAS  PubMed  PubMed Central  Article  Google Scholar 

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

    CAS  PubMed  Article  Google Scholar 

  52. Headd, J. J. et al. Use of knowledge-based restraints in phenix.refine to improve macromolecular refinement at low resolution. Acta Crystallogr. D 68, 381–390 (2012)

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  53. Moriarty, N. W., Grosse-Kunstleve, R. W. & Adams, P. D. electronic Ligand Builder and Optimization Workbench (eLBOW): a tool for ligand coordinate and restraint generation. Acta Crystallogr. D 65, 1074–1080 (2009)

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  54. Tronrud, D. E., Berkholz, D. S. & Karplus, P. A. Using a conformation-dependent stereochemical library improves crystallographic refinement of proteins. Acta Crystallogr. D 66, 834–842 (2010)

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  55. Urzhumtseva, L., Afonine, P. V., Adams, P. D. & Urzhumtsev, A. Crystallographic model quality at a glance. Acta Crystallogr. D 65, 297–300 (2009)

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  56. McCoy, A. J. et al. Phaser crystallographic software. J. Appl. Crystallogr. 40, 658–674 (2007)

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  57. McCoy, A. J., Grosse-Kunstleve, R. W., Storoni, L. C. & Read, R. J. Likelihood-enhanced fast translation functions. Acta Crystallogr. D 61, 458–464 (2005)

    PubMed  Article  CAS  Google Scholar 

  58. Read, R. J. Pushing the boundaries of molecular replacement with maximum likelihood. Acta Crystallogr. D 57, 1373–1382 (2001)

    CAS  PubMed  Article  Google Scholar 

  59. Storoni, L. C., McCoy, A. J. & Read, R. J. Likelihood-enhanced fast rotation functions. Acta Crystallogr. D 60, 432–438 (2004)

    PubMed  Article  CAS  Google Scholar 

  60. Schrodinger, LLC. The PyMOL Molecular Graphics System, Version 1.3r1 (2010)

  61. Helm, I., Jalukse, L., Vilbaste, M. & Leito, I. Micro-Winkler titration method for dissolved oxygen concentration measurement. Anal. Chim. Acta 648, 167–173 (2009)

    CAS  PubMed  Article  Google Scholar 

  62. Ryle, M. J., Padmakumar, R. & Hausinger, R. P. Stopped-flow kinetic analysis of Escherichia coli taurine/α-ketoglutarate dioxygenase: interactions with α-ketoglutarate, taurine, and oxygen. Biochemistry 38, 15278–15286 (1999)

    CAS  PubMed  Article  Google Scholar 

  63. Baldwin, J. et al. Mechanism of rapid electron transfer during oxygen activation in the R2 subunit of Escherichia coli ribonucleotide reductase. 1. Evidence for a transient tryptophan radical. J. Am. Chem. Soc. 122, 12195–12206 (2000)

    CAS  Article  Google Scholar 

Download references

Acknowledgements

We thank H.-w. Liu, S. Elliott and A. Liu for comments on the manuscript. We also thank R. Fan and J. Lee for assistance with the pre-steady state kinetics studies, J. Caradonna for use of stopped-flow instruments, and A. Monzingo for assistance with crystallography software. This work is supported in part by grants from the National Institutes of Health (R01 GM093903 to P.L.; P41 GM104603 to C.E.C.; R01 GM104896 to Y.J.Z.; and R01 GM077387 to M.P.H.), the National Science Foundation (CHE-1309148 to P.L.; CHE-1126268 for the EPR spectrometer), the Welch Foundation (F-1778 to Y.J.Z.), the 973 program (2013CB734000 to L.Z), and Y.G. acknowledges financial support from Carnegie Mellon University. Crystallographic data collection was conducted at advanced light sources (Beamline 5.0.3) and advanced photon sources (BL23-ID-B), Department of Energy (DOE) National User Facility. L.Z. is an awardee of the National Distinguished Young Scholar Program in China (31125002).

Author information

Authors and Affiliations

Authors

Contributions

P.L., Y.J.Z. and L.Z. designed the study. W.Y. conducted the crystallization experiments and structure determination. H.S., F.S., A.S.H., S.W. and N. N. conducted the biochemical studies. C.-H.W., Y.P. and C.E.C. performed the MS–MS analyses. H.S., Y.G., M.P.H. and A.W. conducted the pre-steady state kinetics and EPR characterization. The manuscript was written by P.L., Y.J.Z. and L.Z. with input from all contributing authors.

Corresponding authors

Correspondence to Lixin Zhang, Pinghua Liu or Yan Jessie Zhang.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 Characterization of FtmOx1–α-KG complex.

a, Wild-type FtmOx1 and α-KG binding curve. The increase in absorbance at 520 nm as a function of α-KG concentration when it was added to a solution of wild-type FtmOx1 (0.9 mM) and Feii (0.72 mM) is plotted. On the basis of the equations described in the Methods (determining the α-KG dissociation constant), the Kd for wild-type FtmOx1 and α-KG is 185 ± 35 μM. b, Y224F-substituted FtmOx1 and α-KG binding curve. The increase of absorbance at 520 nm as a function of α-KG concentration when it was added to a solution of Y224F-substituted FtmOx1 (0.9 mM) and Feii (0.7 mM) is plotted. Kd for Y224F-substituted FtmOx1 and α-KG is 198 ± 58 μM. c, Y224A-substituted FtmOx1 and α-KG binding curve. The increase of absorbance at 520 nm as a function of α-KG concentration when it was added to a solution of Y224A-substituted FtmOx1 (0.7 mM) and Feii (0.51 mM) is plotted. Kd for Y224A-substituted FtmOx1 and α-KG is 204 ± 43 μM. In ac, the Kd was calculated based on the concentration of iron-loaded FtmOx1. The experiments were replicated three times and error bars represent s.e.m.

Extended Data Figure 2 Suppression of DOPA formation by the presence of substrate fumitremorgin B.

There is no immediate evidence for the formation of DOPA upon the exposure of the FtmOx1–α-KG complex to O2 when the substrate fumitremorgin B is present. Spectra were recorded after FtmOx1 was used as the control to blank the UV–visible absorption reading.

Extended Data Figure 3 HPLC chromatograms of the FtmOx1 reaction enzyme-concentration dependence.

Chromatograms of FtmOx1 reactions with increasing amounts of FtmOx1 relative to the amount of substrate. The reaction mixture contained 100 mM Tris-HCl, (pH 7.5), 180 μM fumitremorgin B, 2 mM α-ketoglutarate, and variable amounts of FmOx1. Identities of the peaks were assigned based on subsequent NMR and MS characterizations of the isolated compounds. This experiment indicates that FtmOx1 is capable of catalysing endoperoxides formation in the absence of any other reductants.

Extended Data Figure 4 Stoichiometry determination for α-KG and O2 in FtmOx1 reaction.

a, b, Equivalents of endoperoxide products (2 and 3) produced as a function of the ratio of α-KG to iron-loaded FtmOx1 (a) and oxygen to iron-loaded FtmOx1 (b). The quantification was conducted based on the fumitremorgin B (1), compound 2, and compound 3 internal standards. All calculations were based on the concentration of iron-loaded FtmOx1. The experiments were replicated three times and error bars represent s.d.

Extended Data Figure 5 Structural comparison of the active site topologies between FtmOx1 and TauD.

a, Examination of the alternative configuration of α-KG in the FtmOx1–α-KG binary complex using the configuration of α-KG in the TauD–α-KG binary complex. We modelled α-KG in this alternative binding mode and calculated the difference map. In the Fo − Fc map, strong positive density (green) and negative density (red) are shown even when contoured to high level (3.3σ), indicating that this configuration is not correct for the FtmOx1–α-KG complex. b, The Fo − Fc map at the active site of the FtmOx1–fumitremorgin-B complex. A model of the substrate fumitremorgin B is superimposed onto the difference map, which is contoured at 2.8σ. c, Side-by-side comparison of FtmOx1 and TauD active-site topologies. In the left panel, the superimposition of the binary structures of FtmOx1–α-KG and FtmOx1–fumitremorgin-B (1) show that the remaining site for oxygen binding and activation is blocked from the substrate by Y224. d, In contrast, in the structure of the TauD–taurine–α-KG tertiary complex, the remaining site for O2 binding and activation directly faces the substrate (taurine).

Extended Data Figure 6 Characterization of FtmOx1 Y224F variant.

a, Self-hydroxylation reaction in Y224F-substituted FtmOx1. Formation of DOPA upon exposure of the Y224F-substituted FtmOx1–α-KG complex to O2. be, MS/MS analyses of Y224F-substituted FtmOx1. b, MS/MS spectrum of the triply charged parent ion at m/z 768.4109 of a tryptic digested peptide (residue 219–237) from wild-type FtmOx1. c, MS/MS spectrum of the triply charged parent ion at m/z 763.0793 of a tryptic digested peptide (residue 219–237) from Y224F-substituted FtmOx1. d, MS/MS spectrum of the triply charged parent ion at m/z 768.4109 of a tryptic digested peptide (residue 219–237) after exposure Y224F(FtmOx1)–α-KG tertiary complex to O2. e, MS/MS spectrum of the triply charged parent ion at m/z 773.7426 of a tryptic digested peptide (residue 219–237) for DOPA formed upon exposure of FtmOx(Y224F)–α-KG complex to O2 in the absence substrate fumitremorgin B.

Extended Data Figure 7 Mechanistic model for the production of dealkylation products in FtmOx1 Y224A or Y224F variants.

Extended Data Figure 8 Pre-steady-state analyses of FtmOx1 reactions.

a, HPLC chromatograms for FtmOx1 reactions chemically quenched at the indicated times. The reaction mixture in 100 mM Tris-HCl (pH 7.5) buffer contained FtmOx1 (0.65 mM), Feii (0.58 mM), α-KG (12 mM), substrate (0.58 mM), and 20% glycerol. The mixture was mixed with O2-saturated buffer to initiate the reaction. There is an extra signal next to compound 3, which might be due to other chemicals released during the quench process. Results from the chemical quench experiment indicate that FtmOx1 catalysis is on the timescale of a few seconds per cycle. b, Time-dependent 420 nm absorption change (black solid curve) determined by stopped-flow optical absorption spectroscopy and the concentrations of the high-spin Fe3+ species (blue squares) and the g = 2 species (red dots) determined in the rapid-freeze-quench EPR experiments. The black solid curve is associated with the left y axis and is from the average of two stopped-flow trials. The blue squares and red dots are associated with the right y axis and are from the average of two rapid-freeze-quench EPR experiments. The experiments were repeated twice, and error bars reflect the uncertainty of the packing factor of rapid-freeze-quench EPR samples, which is around ±10%.

Extended Data Figure 9 EPR spectroscopic analyses of FtmOx1 reactions.

a, X-band EPR spectra measured at 19 K in reaction samples prepared at the indicated times. The black line shows the sample containing the FtmOx1–Feii–α-KG complex in the absence of O2. (There is a very small signal at g ≈ 4.3 region, only accounted for by <5 μM iron in the sample, which might be due to a very small amount of Fe3+ from inactive enzyme.) Bottom, the reaction sample freeze-quenched at ~0.2 s after mixing the FtmOx1–Feii–α-KG complex with O2. It has two signals: an Fe3+ (g = 4.54, 4.26, and 3.93) and a radical signal at the g = 2 region. b, X-band EPR spectra measured at 19 K for samples freeze-quenched at the indicated times showing the formation of high-spin ferric species on the time scale within 1 s. The reaction was initiated by mixing the FtmOx1–Feii–α-KG complex with O2. g-values are indicated in the figure. c, X-band EPR spectra measured at 19 K for samples freeze-quenched at 0.05 s and the spectral simulation for an S = 5/2 high-spin ferric species. The simulation parameters are: D = 0.3 cm−1, E/D = 0.266, σ(E/D) = 0.03, and g = 4.54, 4.26, 3.93. Measurement conditions in ac: microwave frequency, 9.64 GHz; microwave power, 0.2 mW; modulation amplitude, 1 mT; and modulation frequency, 100 kHz.

Extended Data Table 1 X-ray crystallography data collection and refinement statistics

Supplementary information

Supplementary Information

This file contains Supplementary Figures and Data. (PDF 1613 kb)

FtmOx1 binding to α-KG and substrate

When α-KG is bound to iron center, its configuration in coordinating iron leaves only one open site for water/O2 binding. But this site is blocked from substrate access by Y224. (MOV 16990 kb)

TauD binding to α-KG and substrate.

Unlike FtmOx1, α-KG associates with TauD in a different coordination. The open site for water molecule binding faces directly to the substrate binding pocket. (MOV 17087 kb)

PowerPoint slides

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Yan, W., Song, H., Song, F. et al. RETRACTED ARTICLE: Endoperoxide formation by an α-ketoglutarate-dependent mononuclear non-haem iron enzyme. Nature 527, 539–543 (2015). https://doi.org/10.1038/nature15519

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nature15519

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