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The catalytic pathway of horseradish peroxidase at high resolution

Abstract

A molecular description of oxygen and peroxide activation in biological systems is difficult, because electrons liberated during X-ray data collection reduce the active centres of redox enzymes catalysing these reactions1,2,3,4,5. Here we describe an effective strategy to obtain crystal structures for high-valency redox intermediates and present a three-dimensional movie of the X-ray-driven catalytic reduction of a bound dioxygen species in horseradish peroxidase (HRP). We also describe separate experiments in which high-resolution structures could be obtained for all five oxidation states of HRP, showing such structures with preserved redox states for the first time.

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Figure 1
Figure 2: X-ray-induced reduction of horseradish peroxidase (wavelength range, 0.93–0.98 Å).
Figure 3: X-ray-driven catalytic conversion of a dioxygen species in horseradish peroxidase.
Figure 4: Spectra and refined high-resolution structures for the five oxidation states of horseradish peroxidase.

References

  1. Chance, B., Angiolillo, P., Yang, E. K. & Powers, L. Identification and assay of synchrotron radiation-induced alterations on metalloenzymes and proteins. FEBS Lett. 112, 178–182 (1980)

    CAS  Article  Google Scholar 

  2. Logan, D. T. et al. Crystal structure of reduced protein R2 of ribonucleotide reductase: the structural basis for oxygen activation at a dinuclear iron site. Structure. 4, 1053–1064 (1996)

    CAS  Article  Google Scholar 

  3. Harrenga, A. & Michel, H. The cytochrome c oxidase from Paracoccus denitrificans does not change the metal center ligation upon reduction. J. Biol. Chem. 274, 33296–33299 (1999)

    CAS  Article  Google Scholar 

  4. Schlichting, I. et al. The catalytic pathway of cytochrome p450cam at atomic resolution. Science 287, 1615–1622 (2000)

    ADS  CAS  Article  Google Scholar 

  5. Sjögren, T. & Hajdu, J. Structure of the bound dioxygen species in the cytochrome oxidase reaction of cytochrome cd1 nitrite reductase. J. Biol. Chem. 276, 13072–13076 (2001)

    Article  Google Scholar 

  6. Planche, L. A. Note sur la sophistication de la résine de jalap et sur les moyens de la reconnaître. Bull. Pharmacie 2, 578–580 (1810)

    Google Scholar 

  7. Dunford, H. B. Heme Peroxidases (Wiley, New York, 1999)

    Google Scholar 

  8. Veitch, N. C. & Smith, A. T. Advances in Inorganic Chemistry Vol. 51, 107–162 (Academic, New York, 2001)

    Google Scholar 

  9. Weik, M. et al. Specific chemical and structural damage to proteins produced by synchrotron radiation. Proc. Natl Acad. Sci. USA 97, 623–628 (2000)

    ADS  CAS  Article  Google Scholar 

  10. Burmeister, W. P. Structural changes in a cryo-cooled protein crystal owing to radiation damage. Acta Crystallogr. D 56, 328–341 (2000)

    CAS  Article  Google Scholar 

  11. Leiros, H.-K. S., McSweeney, S. M. & Smalås, A. O. Atomic resolution structures of trypsin provide insight into structural radiation damage. Acta Crystallogr. D 57, 488–497 (2001)

    CAS  Article  Google Scholar 

  12. Lancaster, C. R., Kroger, A., Auer, M. & Michel, H. Structure of fumarate reductase from Wolinella succinogenes at 2.2 Å resolution. Nature 402, 377–385 (1999)

    ADS  CAS  Article  Google Scholar 

  13. Henderson, R. Cryoprotection of protein crystals against radiation-damage in electron and X-ray diffraction. Proc. R. Soc. Lond. B, 241, 6–8 (1990)

    ADS  CAS  Article  Google Scholar 

  14. Henderson, R. The potential and limitations of neutrons, electrons and X-rays for atomic resolution microscopy of unstained biological molecules. Q. Rev. Biophys. 28, 171–193 (1995)

    CAS  Article  Google Scholar 

  15. Gonzales, A. & Nave, C. Radiation damage in protein crystals at low temperature. Acta Crystallogr. D 50, 874–877 (1994)

    Article  Google Scholar 

  16. Neutze, R., Wouts, R., van der Spoel, D., Weckert, E. & Hajdu, J. Potential for biomolecular imaging with femtosecond X-ray pulses. Nature 406, 752–757 (2000)

    ADS  CAS  Article  Google Scholar 

  17. Ziaja, B., van der Spoel, D., Szöke, A. & Hajdu, J. Auger-electron cascades in diamond and amorphous carbon. Phys. Rev. B 64, 214104-1–214104-8 (2001)

    ADS  Article  Google Scholar 

  18. Persson, P., Lunell, S., Szöke, A., Ziaja, B. & Hajdu, J. Shake-up and shake-off excitations with associated electron losses in X-ray studies of proteins. Protein Sci. 10, 2480–2484 (2001)

    CAS  Article  Google Scholar 

  19. Nakajima, R. & Yamazaki, I. The mechanism of oxyperoxidase formation from ferryl peroxidase and hydrogen peroxide. J. Biol. Chem. 262, 2576–2581 (1987)

    CAS  PubMed  Google Scholar 

  20. Van Wart, H. E. & Zimmer, J. Resonance Raman evidence for the activation of dioxygen in horseradish oxyperoxidase. J. Biol. Chem. 260, 8372–8377 (1985)

    CAS  PubMed  Google Scholar 

  21. Hammes-Schiffer, S. Theoretical perspectives on proton-coupled electron transfer reactions. Acc. Chem. Res. 34, 273–281 (2001)

    CAS  Article  Google Scholar 

  22. Schlichting, I., Berendzen, J., Phillips, G. N. Jr & Sweet, R. M. Crystal structure of photolysed carbonmonoxy-myoglobin. Nature 371, 808–812 (1994)

    ADS  CAS  Article  Google Scholar 

  23. Edman, K. et al. High-resolution X-ray structure of an early intermediate in the bacteriorhodopsin photocycle. Nature 401, 822–826 (1999)

    ADS  CAS  Article  Google Scholar 

  24. Gajhede, M. et al. Crystal structure of horseradish peroxidase C at 2.15 angstrom resolution. Nature Struct. Biol. 4, 1032–1038 (1997)

    CAS  Article  Google Scholar 

  25. Chance, B. et al. X-ray absorption studies of intermediates in peroxidase activity. Arch. Biochem. Biophys. 235, 596–611 (1984)

    CAS  Article  Google Scholar 

  26. Szöke, A. Use of statistical information in X-ray crystallography with application to the holographic method. Acta Crystallogr. A 54, 543–562 (2001)

    Article  Google Scholar 

  27. Cravens, T. E. Comet Hyakutake X-ray source: Charge transfer of solar wind heavy ions. Geophys. Res. Lett. 24, 105–108 (1997)

    ADS  CAS  Article  Google Scholar 

  28. Henriksen, A., Smith, A. T. & Gajhede, M. The structures of the horseradish peroxidase C-ferulic acid complex and the ternary complex with cyanide suggest how peroxidases oxidize small phenolic substrates. J. Biol. Chem. 274, 35005–35011 (1999)

    CAS  Article  Google Scholar 

  29. Smith, A. T. et al. Expression of a synthetic gene for horseradish peroxidase C in Escherichia coli and folding and activation of the recombinant enzyme with calcium and heme. J. Biol. Chem. 265, 13335–13343 (1990)

    CAS  PubMed  Google Scholar 

  30. Read, R. J. Improved Fourier coefficients for maps using phases from partial structures with errors. Acta Crystallogr. A 42, 140–149 (1986)

    Article  Google Scholar 

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Acknowledgements

We thank M. Gajhede, K. G. Welinder, N. Veitch, B. Ziaja, D. Choudhury, M. Iwata, R. Subramanian and G. Katona for discussions and help. We thank MaxLab and ESRF/EMBO for beam time and services. This work was supported by the EU-Biotech Programme and by the Swedish Research Councils.

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Correspondence to Janos Hajdu.

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Berglund, G., Carlsson, G., Smith, A. et al. The catalytic pathway of horseradish peroxidase at high resolution. Nature 417, 463–468 (2002). https://doi.org/10.1038/417463a

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