Detection and Characterization of Heme-Thiolate Compound II from AaeAPO PeroxygenaseOpen image in new window

  • Xiaoshi WangEmail author
Part of the Springer Theses book series (Springer Theses)


A kinetic and spectroscopic characterization of the unusual, high-valent Cys–FeIV–OH intermediate (compound II) from AaeAPO, the heme-thiolate peroxygenase from Agrocybe aegerita, is described. AaeAPO-II was generated by the reaction of ferric enzyme with mCPBA in the presence of nitroxides and detected with the use of fast-mixing UV-vis stopped-flow spectroscopy. The nitroxides served as a selective reductant of AaeAPO-I, reacting only slowly with AaeAPO-II. AaeAPO-II displayed a characteristic split Soret UV-vis spectrum (370 and 428 nm). Rapid-mixing, pH-jump spectrophotometry revealed a basic pK a of 10 for the FeIV–O–H of AaeAPO-II, indicating that AaeAPO-II is protonated under typical turnover conditions. Kinetic characterization showed that AaeAPO-II is unusually reactive toward a panel of benzylic C–H and phenolic substrates, with second-order rate constants in the range of 10–107 M−1 s−1. Our results demonstrate the important role of the axial cysteine ligand in increasing the proton affinity of the Fe-oxo, AaeAPO compound I, thus providing driving force of C–H bonds oxygenation.


Phenol Oxidation Kinetic Characterization Nitrosyl Complex Hydrogen Atom Abstraction Cyclohexane Carboxylic 
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  1. 1.
    Austin, R.N., Groves, J.T.: Alkane-oxidizing metalloenzymes in the carbon cycle. Metallomics 3, 775–787 (2011)CrossRefGoogle Scholar
  2. 2.
    Lewis, J.C., Coelho, P.S., Arnold, F.H.: Enzymatic functionalization of carbon-hydrogen bonds. Chem. Soc. Rev. 40, 2003–2021 (2011)CrossRefGoogle Scholar
  3. 3.
    Groves, J.T.: High-valent iron in chemical and biological oxidations. J. Inorg. Biochem. 100, 434–447 (2006)CrossRefGoogle Scholar
  4. 4.
    Ortiz de Montellano, P.R.: Hydrocarbon hydroxylation by cytochrome P450 enzymes. Chem. Rev. 110, 932–948 (2010)Google Scholar
  5. 5.
    Groves, J.T.: Models and Mechanisms of Cytochrome P-450 Action Chap. 1 in, 3e, pp. 1–44. Kluwer Academic/Plenum Publishers, New York (2004). In: Ortiz de Montellano, P.R. (ed.) Cytochrome P450: Structure, Mechanism, and Biochemistry, 3 edn, pp. 1–44. Kluwer Academic/Plenum, New York (2005)Google Scholar
  6. 6.
    Guengerich, F.P.: Cytochrome P450: what have we learned and what are the future issues? Durg Metab. Rev. 36, 159–197 (2004)CrossRefGoogle Scholar
  7. 7.
    Luthra, A., Denisov, I.G., Sligar, S.G.: Spectroscopic features of cytochrome P450 reaction intermediates. Arch. Biochem. Biophys. 507, 26–35 (2011)CrossRefGoogle Scholar
  8. 8.
    Green, M.T.: C{single bond}H bond activation in heme proteins: the role of thiolate ligation in cytochrome P450. Curr. Op. Chem. Biol. 13, 84–88 (2009)CrossRefGoogle Scholar
  9. 9.
    Hrycay, E.G., Bandiera, S.M.: The monooxygenase, peroxidase, and peroxygenase properties of cytochrome P450. Arch. Biochem. Biophys. 522, 71–89 (2012)CrossRefGoogle Scholar
  10. 10.
    Shaik, S., Lai, W., Chen, H., Wang, Y.: The valence bond way: reactivity patterns of cytochrome P450 enzymes and synthetic analogs. Acc. Chem. Res. 43, 1154–1165 (2010)CrossRefGoogle Scholar
  11. 11.
    Jung, C., Vries, S.D., Schünemann, V.: Spectroscopic characterization of cytochrome P450 Compound I. Arch. Biochem. Biophys. 507, 44–55 (2011)CrossRefGoogle Scholar
  12. 12.
    Jung, C.: The mystery of cytochrome P450 compound I: a mini-review dedicated to Klaus Ruckpaul. BBA-Proteins Proteom. 1814, 46–57 (2011)CrossRefGoogle Scholar
  13. 13.
    Rittle, J., Green, M.T.: Cytochrome P450 compound I: capture, characterization, and C-H bond activation kinetics. Science 330, 933–937 (2010)CrossRefGoogle Scholar
  14. 14.
    Behan, R.K., Hoffart, L.M., Stone, K.L., Krebs, C., Green, M.T.: Evidence for basic ferryls in cytochromes P450. J. Am. Chem. Soc. 128, 11471–11474 (2006)CrossRefGoogle Scholar
  15. 15.
    Stone, K.L., Behan, R.K., Green, M.T.: X-ray absorption spectroscopy of chloroperoxidase compound I: insight into the reactive intermediate of P450 chemistry. Proc. Natl. Acad. Sci. U.S.A. 102, 16563–16565 (2005)CrossRefGoogle Scholar
  16. 16.
    Green, M.T., Dawson, J.H., Gray, H.B.: Oxoiron(IV) in chloroperoxidase compound II is basic: implications for P450 chemistry. Science 304, 1653–1656 (2004)CrossRefGoogle Scholar
  17. 17.
    Egawa, T., Proshlyakov, D.A., Miki, H., Makino, R., Ogura, T., Kitagawa, T., Ishimura, Y.: Effects of a thiolate axial ligand on the π → π* electronic states of oxoferryl porphyrins: a study of the optical and resonance Raman spectra of compounds I and II of chloroperoxidase. J. Biol. Inorg. Chem. 6, 46–54 (2001)CrossRefGoogle Scholar
  18. 18.
    Bell, S.R., Groves, J.T.: A highly reactive P450 model compound I. J. Am. Chem. Soc. 131, 9640–9641 (2009)CrossRefGoogle Scholar
  19. 19.
    Groves, J.T., Gross, Z., Stern, M.K.: Preparation and reactivity of oxoiron(IV) porphyrins. Inorg. Chem. 33, 5065–5072 (1994)CrossRefGoogle Scholar
  20. 20.
    Ullrich, R., Nüske, J., Scheibner, K., Spantzel, J., Hofrichter, M.: Novel haloperoxidase from the agaric basidiomycete Agrocybe aegerita oxidizes aryl alcohols and aldehydes. Appl. Environ. Microb. 70, 4575–4581 (2004)CrossRefGoogle Scholar
  21. 21.
    Piontek, K., Ullrich, R., Liers, C., Diederichs, K., Plattner, D.A., Hofrichter, M.: Crystallization of a 45 kDa peroxygenase/peroxidase from the mushroom Agrocybe aegerita and structure determination by SAD utilizing only the haem iron. Acta Crystallogr. F 66, 693–698 (2010)CrossRefGoogle Scholar
  22. 22.
    Pecyna, M.J., Ullrich, R., Bittner, B., Clemens, A., Scheibner, K., Schubert, R., Hofrichter, M.: Molecular characterization of aromatic peroxygenase from Agrocybe aegerita. Appl. Microbiol. Biotech. 84, 885–897 (2009)CrossRefGoogle Scholar
  23. 23.
    Peter, S., Kinne, M., Wang, X., Ullrich, R., Kayser, G., Groves, J.T., Hofrichter, M.: Selective hydroxylation of alkanes by an extracellular fungal peroxygenase. FEBS J. 278, 3667–3675 (2011)CrossRefGoogle Scholar
  24. 24.
    Hofrichter, M., Ullrich, R., Pecyna, M.J., Liers, C., Lundell, T.: New and classic families of secreted fungal heme peroxidases. Appl. Microbiol. Biotechn. 87, 871–897 (2010)CrossRefGoogle Scholar
  25. 25.
    Poraj-Kobielska, M., Kinne, M., Ullrich, R., Scheibner, K., Kayser, G., Hammel, K.E., Hofrichter, M.: Preparation of human drug metabolites using fungal peroxygenases. Biochem. Pharmacol. 82, 789–796 (2011)CrossRefGoogle Scholar
  26. 26.
    Wang, X., Peter, S., Kinne, M., Hofrichter, M., Groves, J.T.: Detection and kinetic characterization of a highly reactive heme-thiolate peroxygenase compound I. J. Am. Chem. Soc. 134, 12897–12900 (2012)CrossRefGoogle Scholar
  27. 27.
    Wang, X., Peter, S., Ullrich, R., Hofrichter, M., Groves, J.T.: Driving force for oxygen-atom transfer by heme-thiolate enzymes. Angew. Chem. Int. Ed. (2013)Google Scholar
  28. 28.
    Blinco, J.P., Hodgson, J.L., Morrow, B.J., Walker, J.R., Will, G.D., Coote, M.L., Bottle, S.E.: Experimental and theoretical studies of the redox potentials of cyclic nitroxides. J. Org. Chem. 73, 6763–6771 (2008)CrossRefGoogle Scholar
  29. 29.
    Lambeir, A.M., Dunford, H.B.: A kinetic and spectral study of the alkaline transitions of chloroperoxidase. Arch. Biochem. Biophys. 220, 549–556 (1983)CrossRefGoogle Scholar
  30. 30.
    Stone, K.L., Behan, R.K., Green, M.T.: Resonance Raman spectroscopy of chloroperoxidase compound II provides direct evidence for the existence of an iron(IV)-hydroxide. Proc. Natl. Acad. Sci. U.S.A. 103, 12307–12310 (2006)CrossRefGoogle Scholar
  31. 31.
    Green, M.T.: Application of Badger’s rule to heme and non-heme iron-oxygen bonds: an examination of ferryl protonation states. J. Am. Chem. Soc. 128, 1902–1906 (2006)CrossRefGoogle Scholar
  32. 32.
    Mayer, J.M.: Hydrogen atom abstraction by metal-oxo complexes: understanding the analogy with organic radical reactions. Acc. Chem. Res. 31, 441–450 (1998)CrossRefGoogle Scholar
  33. 33.
    Newcomb, M., Halgrimson, J.A., Horner, J.H., Wasinger, E.C., Chen, L.X., Sligar, S.G.: X-ray absorption spectroscopic characterization of a cytochrome P450 compound II derivative. Proc. Natl. Acad. Sci. U.S.A. 105, 8179–8184 (2008)CrossRefGoogle Scholar
  34. 34.
    Yuan, X., Sheng, X., Horner, J.H., Bennett, B., Fung, L.W.M., Newcomb, M.: Low temperature photo-oxidation of chloroperoxidase compound II. J. Inorg. Biochem. 104, 1156–1163 (2010)CrossRefGoogle Scholar
  35. 35.
    Behan, R.K., Hoffart, L.M., Stone, K.L., Krebs, C., Green, M.T.: Reaction of cytochrome P450(BM3) and peroxynitrite yields nitrosyl complex. J. Am. Chem. Soc. 129, 5855–5859 (2007)CrossRefGoogle Scholar
  36. 36.
    Lambeir, A.M., Dunford, H.B., Pickard, M.A.: Kinetics of the oxidation of ascorbic acid, ferrocyanide and p-phenolsulfonic acid by chloroperoxidase compounds I and II. Eur. J. Biochem. 163, 123–127 (1987)CrossRefGoogle Scholar
  37. 37.
    Sakurada, J., Sekiguchi, R., Sato, K., Hosoya, T.: Kinetic and molecular orbital studies on the rate of oxidation of monosubstituted phenols and anilines by horseradish peroxidase compound II. Biochemistry 29, 4093–4098 (1990)CrossRefGoogle Scholar
  38. 38.
    Dunford, H.B., Adeniran, A.J.: Hammett ρ{variant}σ correlation for reactions of horseradish peroxidase compound II with phenols. Arch. Biochem. Biophys. 251, 536–542 (1986)CrossRefGoogle Scholar
  39. 39.
    Osborne, R.L., Coggins, M.K., Terner, J., Dawson, J.H.: Caldariomyces fumago chloroperoxidase catalyzes the oxidative dehalogenation of chlorophenols by a mechanism involving two one-electron steps. J. Am. Chem. Soc. 129, 14838–14839 (2007)CrossRefGoogle Scholar
  40. 40.
    Nam, W., Park, S.E., Lim, I.K., Lim, M.H., Hong, J., Kim, J.: First direct evidence for stereospecific olefin epoxidation and alkane hydroxylation by an oxoiron(IV) porphyrin complex. J. Am. Chem. Soc. 125, 14674–14675 (2003)CrossRefGoogle Scholar
  41. 41.
    Pan, Z., Newcomb, M.: Kinetics and mechanism of oxidation reactions of porphyrin-iron(IV)-oxo intermediates. Inorg. Chem. 46, 6767–6774 (2007)CrossRefGoogle Scholar
  42. 42.
    Colclough, N., Lindsay Smith, J.R.: A mechanistic study of the oxidation of phenols in aqueous solution by oxoiron(IV) tetra(N-methylpyridyl)porphyrins. A model for horseradish peroxidase compound II? J. Chem. Soc. Perkin Trans. 2, 1139–1149 (1994)Google Scholar
  43. 43.
    Bell, S.R.: Modelling Heme Monooxygenases with Water-Soluble Iron Porphyrins. Princeton University (2010)Google Scholar
  44. 44.
    Gardner, K.A., Kuehnert, L.L., Mayer, J.M.: Hydrogen atom abstraction by permanganate: oxidations of arylalkanes in organic solvents. Inorg. Chem. 36, 2069–2078 (1997)CrossRefGoogle Scholar
  45. 45.
    Whitehouse, C.J.C., Bell, S.G., Wong, L.-L.: Desaturation of alkylbenzenes by cytochrome P450BM3 (CYP102A1). Chem. Eur. J. 14, 10905–10908 (2008)CrossRefGoogle Scholar
  46. 46.
    Dust, J.M., Arnold, D.R.: Substituent effects on benzyl radical ESR hyperfine coupling constants. The σα· scale based upon spin delocalization. J. Am. Chem. Soc. 105, 1221–1227 (1983)CrossRefGoogle Scholar
  47. 47.
    Krygowski, T.M., Stȩpień, B.T.: Sigma- and Pi-electron delocalization: focus on substituent effects. Chem. Rev. 105, 3482–3512 (2005)CrossRefGoogle Scholar
  48. 48.
    Das, P.K., Encinas, M.V., Steenken, S., Scaiano, J.C.: Reaction of tert-butoxy radicals with phenols. Comparison with the reactions of carbonyl triplets. J. Am. Chem. Soc. 103, 4162–4166 (1981)CrossRefGoogle Scholar
  49. 49.
    Howard, J.A., Ingold, K.U., Symonds, M.: Absolute rate constants for hydrocarbon oxidation 8. Reactions of cumylperoxy radicals. Can. J. Chem. 46, 1017–000 (1968)CrossRefGoogle Scholar
  50. 50.
    Nahor, G.S., Neta, P., Alfassi, Z.B.: Perfluorobutylperoxyl radical as an oxidant in various solvents. J. Phys. Chem. 95, 4419–4422 (1991)CrossRefGoogle Scholar
  51. 51.
    Powell, M.F., Wu, J.C., Bruice, T.C.: Ferricyanide oxidation of dihydropyridines and analogs. J. Am. Chem. Soc. 106, 3850–3856 (1984)CrossRefGoogle Scholar
  52. 52.
    Sapkota, K., Roh, E., Lee, E., Ha, E.M., Yang, J.H., Lee, E.S., Kwon, Y., Kim, Y., Na, Y.: Synthesis and anti-melanogenic activity of hydroxyphenyl benzyl ether analogues. Bioorg. Med. Chem. 19, 2168–2175 (2011)CrossRefGoogle Scholar

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© Springer International Publishing Switzerland 2016

Authors and Affiliations

  1. 1.PhiladelphiaUSA

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