Mechanistic insight into the initiation step of the reaction of Burkholderia pseudomallei catalase-peroxidase with peroxyacetic acid

Original Paper


The reaction of the catalase-peroxidase of Burkholderia pseudomallei with peroxyacetic acid has been analyzed using stopped-flow spectrophotometry. Two well-defined species were observed, the first defined by an increase in intensity and narrowing of the Soret band at 407 nm and a 10-nm shift of the charge transfer band from 635 to 625 nm. These features are consistent with a ferric spectrum with a greater proportion of sixth-coordination character and are assigned to an FeIII–peroxyacetic acid complex. Complementary 9-GHz EPR characterization of the changes in the ferric signal of the resting enzyme induced by the binding of acetate in the heme pocket substantiates the proposal. Kinetic analysis of the spectral changes as a function of peroxyacetic acid concentration revealed two independent peroxyacetic acid binding events, one coincident with formation of the FeIII–peroxyacetic acid complex and the other coincident with the heme oxidation to the subsequent ferryl intermediate. A model to explain the need for two peroxyacetic acid binding events is proposed. The reaction of the W330F variant followed similar kinetics, although the characteristic spectral features of the FeIV=O Por•+ species were detected. The variant D141A lacking an aspartate at the entrance to the heme cavity as well as the R108A and D141A/R108A variants showed no evidence for the FeIII–peroxyacetic acid complex, only the formation of ferryl species with absorbance maxima at 414, 545, and 585 nm.


Catalase-peroxidase Enzyme–peroxyacetic acid complex Ferryl heme iron Compound I EPR spectroscopy 



This work was supported by the French CNRS and CEA Saclay (to A.I.), a Ph.D. Fellowship (CFR contract from CEA Saclay to J.C.), and grants from the Natural Sciences and Engineering Research Council of Canada (to P.C.L.) and the Canadian Research Chair Program (to P.C.L.).

Supplementary material

775_2009_493_MOESM1_ESM.pdf (97 kb)
Supplementary material 1 (PDF 98 kb)


  1. 1.
    Singh R, Wiseman B, Deemagarn T, Donald LJ, Duckworth HW, Carpena X, Fita I, Loewen PC (2004) J Biol Chem 279:43098–43106PubMedCrossRefGoogle Scholar
  2. 2.
    Singh R, Wiseman B, Deemagarn T, Jha V, Switala J, Loewen PC (2008) Arch Biochem Biophys 417:207–214CrossRefGoogle Scholar
  3. 3.
    Claiborne A, Fridovich I (1979) J Biol Chem 254:4245–4252PubMedGoogle Scholar
  4. 4.
    Loewen PC, Triggs BL, George CS, Hrabarchuk BE (1985) J Bacteriol 162:661–667PubMedGoogle Scholar
  5. 5.
    Triggs-Raine BL, Doble BL, Mulvey MR, Sorby PA, Loewen PC (1988) J Bacteriol 170:4415–4419PubMedGoogle Scholar
  6. 6.
    Zhang Y, Heym B, Allen B, Young D, Cole S (1992) Nature 358:591–593PubMedCrossRefGoogle Scholar
  7. 7.
    Sivaraja M, Goodin DB, Smith M, Hoffman B (1989) Science 245:738–740PubMedCrossRefGoogle Scholar
  8. 8.
    Blodig W, Smith AT, Winterhalter K, Piontek K (1999) Arch Biochem Biophys 370:86–92PubMedCrossRefGoogle Scholar
  9. 9.
    Singh R, Switala J, Loewen PC, Ivancich A (2007) J Am Chem Soc 129:15954–15963PubMedCrossRefGoogle Scholar
  10. 10.
    Chouchane S, Lippai I, Magliozzo RS (2000) Biochemistry 39:9975–9983PubMedCrossRefGoogle Scholar
  11. 11.
    Jakopitsch C, Auer M, Regelsberger G, Jantschko W, Furtmuller PG, Ruker F, Obinger C (2003) Biochemistry 42:5292–5300PubMedCrossRefGoogle Scholar
  12. 12.
    Jakopitsch C, Ivancich A, Schmuckenschlager F, Wanasinghe A, Potl G, Furtmuller PG, Ruker F, Obinger C (2004) J Biol Chem 279:46082–46095PubMedCrossRefGoogle Scholar
  13. 13.
    Jakopitsch C, Auer M, Regelsberger G, Jantschko W, Furtmuller PG, Ruker F, Obinger C (2003) Eur J Biochem 270:1006–1013PubMedCrossRefGoogle Scholar
  14. 14.
    Jakopitsch C, Auer M, Ivancich A, Ruker F, Furtmuller PG, Obinger C (2003) J Biol Chem 278:20185–20191PubMedCrossRefGoogle Scholar
  15. 15.
    Regelsberger G, Jakopitsch D, Ruker F, Krois D, Peschek GA, Obinger C (2000) J Biol Chem 275:22854–22861PubMedCrossRefGoogle Scholar
  16. 16.
    Jakopitsch C, Vlasits J, Wiseman B, Loewen PC, Obinger C (2007) Biochemistry 46:1183–1193PubMedCrossRefGoogle Scholar
  17. 17.
    Ghiladi RA, Knudsen GM, Medzihradszky KF, Ortiz de Montellano PR (2005) J Biol Chem 280:22651–22663PubMedCrossRefGoogle Scholar
  18. 18.
    Spotilak T, Dawson JH, Ballou DP (2005) J Biol Chem 280:20300–20309CrossRefGoogle Scholar
  19. 19.
    Carpena X, Switala J, Loprasert S, Mongkolsuk S, Fita I, Loewen PC (2002) Acta Crystallogr D 58:2184–2186PubMedCrossRefGoogle Scholar
  20. 20.
    Rørth M, Jensen PK (1967) Biochim Biophys Acta 139:171–173PubMedGoogle Scholar
  21. 21.
    Childs RE, Bardsley WG (1975) Biochem J 145:93–103PubMedGoogle Scholar
  22. 22.
    Layne E (1957) Methods Enzymol 3:447–454CrossRefGoogle Scholar
  23. 23.
    Carpena X, Wiseman B, Deemagarn T, Herguedas B, Ivancich A, Singh R, Loewen PC, Fita I (2006) Biochemistry 45:5171–5179PubMedCrossRefGoogle Scholar
  24. 24.
    Emsley P, Cowtan K (2004) Acta Crystallogr D 60:2126–2132PubMedCrossRefGoogle Scholar
  25. 25.
    Deemagarn T, Wiseman B, Carpena X, Ivancich A, Fita I, Loewen PC (2007) Proteins 66:219–228PubMedCrossRefGoogle Scholar
  26. 26.
    Schonbaum GR (1973) J Biol Chem 248:502–511PubMedGoogle Scholar
  27. 27.
    Henriksen A, Schuller DJ, Meno K, Welinder KG, Smith AT, Gajhede M (1998) Biochemisty 37:8054–8060CrossRefGoogle Scholar
  28. 28.
    Loewen PC, Carpena X, Rovira C, Haas R, Odenbreit S, Nicholls P, Fita I (2004) Biochemistry 43:3089–3103PubMedCrossRefGoogle Scholar
  29. 29.
    Dunford HB (1999) Heme peroxidases. Wiley, New YorkGoogle Scholar
  30. 30.
    Fielding AJ, Singh R, Boscolo B, Loewen PC, Ghibaudi EI, Ivancich A (2008) Biochemistry 47:9781–9792PubMedCrossRefGoogle Scholar
  31. 31.
    Ivancich A, Jakopitsch C, Auer M, Un S, Obinger C (2003) J Am Chem Soc 125:14093–14102PubMedCrossRefGoogle Scholar
  32. 32.
    Erman JE, Vitello LB, Mauro JM, Kraut J (1989) Biochemistry 28:7992–7995PubMedCrossRefGoogle Scholar
  33. 33.
    Rodriguez-Lopez JN, Smith AT, Thorneley RNF (1996) J Biol Chem 271:4023–4030PubMedCrossRefGoogle Scholar
  34. 34.
    Loew G, Dupuis M (1996) J Am Chem Soc 118:10584–10587CrossRefGoogle Scholar
  35. 35.
    Derat E, Shaik S, Rovira C, Vidossich P, Alfonso-Prieto M (2007) J Am Chem Soc 129:6346–6347PubMedCrossRefGoogle Scholar

Copyright information

© SBIC 2009

Authors and Affiliations

  1. 1.Department of MicrobiologyUniversity of ManitobaWinnipegCanada
  2. 2.Centre d’Etudes de SaclayIBITEC, Service de Bioénergétique, Biologie Structurale et Mécanismes, CNRS URA 2096Gif-sur-YvetteFrance
  3. 3.Department of Chemistry and Biochemistry, School of Life SciencesUniversity of SussexBrightonUK

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