Hydrocarbon Hydroxylations Catalyzed by AaeAPO: Evidence of Radical Intermediates and Kinetic Isotope EffectsOpen image in new window

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


Recently, a new heme-thiolate peroxygenase enzyme from the fungus Agrocybe aegerita, was identified and found to be capable of catalyzing hydrocarbon hydroxylation by using H2O2 as co-substrate with high efficiency and selectivity. It not only shows potential for practical biocatalytic application but also might provide some inspiration for designing biomimetic catalysts. In order to investigate the reaction mechanism, several radical clocks were used in this chapter. Products indicative of radical intermediates were all detected during the oxidation of norcarane, bicycle[2.1.0]pentane and 1,1,2,2-tetremethylcycloprotane with lifetimes ranging from 3.0 to 132 ps. At the same time, a large intramolecular deuterium isotope effect was measured with the hydroxylation of 1,1,1,2,2,3,3-d 7-n-hexane and methyl partially deuterated toluenes. Taken together, a mechanism involving hydrogen atom abstraction and rebound is suggested. More interestingly, small intramolecular KIEs probed by (R)-1-ethylbenzene were also observed. We thought these suppressions were caused by steric hindrance in the active site of enzyme. These isotope masking effects can give us some information about the dynamics of substrates and map the active site environment of enzyme.


Isotope Effect Mandelic Acid Kinetic Isotope Effect Hydrogen Atom Abstraction Desaturation Product 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.


  1. 1.
    Groves, J.T., Mcclusky, G.A., White, R.E., Coon, M.J.: Aliphatic hydroxylation by highly purified liver microsomal cytochrome-P-450—evidence for a carbon radical intermediate. Biochem. Biophys. Res. Co. 81, 154–160 (1978)CrossRefGoogle Scholar
  2. 2.
    Griller, D., Ingold, K.U.: Free-Radical clocks. Acc. Chem. Res. 13, 317–323 (1980)CrossRefGoogle Scholar
  3. 3.
    Ortiz de Montellano, P.R.: Hydrocarbon hydroxylation by cytochrome P450 enzymes. Chem. Rev. 110, 932–948 (2010)CrossRefGoogle Scholar
  4. 4.
    Johnson, K.A.: Transient-state kinetic analysis of enzyme reaction pathways. In: The Enzymes, vol. XX, pp. 1–61 (1992)Google Scholar
  5. 5.
    Newcomb, M., Toy, P.H.: Hypersensitive radical probes and the mechanisms of cytochrome P450-catalyzed hydroxylation reactions. Acc. Chem. Res. 33, 449–455 (2000)CrossRefGoogle Scholar
  6. 6.
    Demontellano, P.R.O., Stearns, R.A.: Timing of the radical recombination step in cytochrome-P-450 catalysis with ring-strained probes. J. Am. Chem. Soc. 109, 3415–3420 (1987)CrossRefGoogle Scholar
  7. 7.
    Auclair, K., Hu, Z.B., Little, D.M., de Montellano, P.R.O., Groves, J.T.: Revisiting the mechanism of P450 enzymes with the radical clocks norcarane and spiro[2,5]octane. J. Am. Chem. Soc. 124, 6020–6027 (2002)CrossRefGoogle Scholar
  8. 8.
    Newcomb, M., Shen, R., Lu, Y., Coon, M.J., Hollenberg, P.F., Kopp, D.A., Lippard, S.J.: Evaluation of norcarane as a probe for radicals in cytochome P450—and soluble methane monooxygenase-catalyzed hydroxylation reactions. J. Am. Chem. Soc. 124, 6879–6886 (2002)CrossRefGoogle Scholar
  9. 9.
    Bertrand, E., Sakai, R., Rozhkova-Novosad, E., Moe, L., Fox, B.G., Groves, J.T., Austin, R.N.: Reaction mechanisms of non-heme diiron hydroxylases characterized in whole cells. J. Inorg. Biochem. 99, 1998–2006 (2005)CrossRefGoogle Scholar
  10. 10.
    Chakrabarty, S., Austin, R.N., Deng, D., Groves, J.T., Lipscomb, J.D.: Radical intermediates in monooxygenase reactions of Rieske dioxygenases. J. Am. Chem. Soc. 129, 3514–3515 (2007)CrossRefGoogle Scholar
  11. 11.
    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
  12. 12.
    Cooper, H.L.R., Groves, J.T.: Molecular probes of the mechanism of cytochrome P450. Oxygen traps a substrate radical intermediate. Arch. Biochem. Biophys. 507, 111–118 (2011)CrossRefGoogle Scholar
  13. 13.
    Austin, R.N., Luddy, K., Erickson, K., Pender-Cudlip, M., Bertrand, E., Deng, D., Buzdygon, R.S., Van Beilen, J.B., Groves, J.T.: Cage escape competes with geminate recombination during alkane hydroxylation by the diiron oxygenase AlkB. Angew. Chem. Int. Ed. 47, 5232–5234 (2008)CrossRefGoogle Scholar
  14. 14.
    Bowry, V.W., Ingold, K.U.: A radical clock investigation of microsomal cytochrome P-450 hydroxylation of hydrocarbons. Rate of oxygen rebound. J. Am. Chem. Soc. 113, 5699–5707 (1991)CrossRefGoogle Scholar
  15. 15.
    Nelson, S.D., Trager, W.F.: The use of deuterium isotope effects to probe the active site properties, mechanism of cytochrome P450-catalyzed reactions, and mechanisms of metabolically dependent toxicity. Drug Metab. Dispos. 31, 1481–1498 (2003)CrossRefGoogle Scholar
  16. 16.
    Hanzlik, R.P., Ling, K.H.J.: Active site dynamics of toluene hydroxylation by cytochrome P-450. J. Org. Chem. 55, 3992–3997 (1990)CrossRefGoogle Scholar
  17. 17.
    Audergon, C., Iyer, K.R., Jones, J.P., Darbyshire, J.F., Trager, W.F.: Experimental and theoretical study of the effect of active-site constrained substrate motion on the magnitude of the observed intramolecular isotope effect for the P450 101 catalyzed benzylic hydroxylation of isomeric xylenes and 4,4’-dimethylbiphenyl. J. Am. Chem. Soc. 121, 41–47 (1999)CrossRefGoogle Scholar
  18. 18.
    Henne, K.R., Fisher, M.B., Iyer, K.R., Lang, D.H., Trager, W.F., Rettie, A.E.: Active site characteristics of CYP4B1 probed with aromatic ligands. Biochemistry 40, 8597–8605 (2001)CrossRefGoogle Scholar
  19. 19.
    Harrelson, J.P., Henne, K.R., Alonso, D.O.V., Nelson, S.D.: A comparison of substrate dynamics in human CYP2E1 and CYP2A6. Biochem. Biophys. Res. Co. 352, 843–849 (2007)CrossRefGoogle Scholar
  20. 20.
    White, R.E., Miller, J.P., Favreau, L.V., Bhattacharyya, A.: Stereochemical dynamics of aliphatic hydroxylation by cytochrome P-450. J. Am. Chem. Soc. 108, 6024–6031 (1986)CrossRefGoogle Scholar
  21. 21.
    Elsenbaumer, R.L., Mosher, H.S.: Enantiomerically pure (R)-(+)-2-phenylethanol-2-d and -1,1,2-d3, and (S)-(+)-1-phenylethane-1-d, -1,2,-d2, -1,2,2-d3, and -1,2,2,2-d4. J. Org. Chem. 44, 600–604 (1979)CrossRefGoogle Scholar
  22. 22.
    Legoff, E.: Cyclopropanes from an easily prepared, highly active zinc-copper couple, dibromomethane, and olefins. J. Org. Chem. 29, 2048–2050 (1964)CrossRefGoogle Scholar
  23. 23.
    Lambert, J.B., Marko, D.E.: Factors influencing conformational preferences in cyclohexenes. J. Am. Chem. Soc. 107, 7978–7982 (1985)CrossRefGoogle Scholar
  24. 24.
    Åkermark, B., Hansson, S., Rein, T., Vgberg, J., Heumann, A., Bäckvall, J.E.: Palladium-catalyzed allylic acetoxylation: an exploratory study of the influence of added acids. J. Orgaomet. Chem. 369, 433–444 (1989)CrossRefGoogle Scholar
  25. 25.
    Snider, B.B., Rodini, D.J.: Diaklylaluminum chloride catalyzed ene reactions of aldehydes. Synth. Ipsenol. Tetrahedron Lett. 21, 1815–1818 (1980)CrossRefGoogle Scholar
  26. 26.
    Gassman, P.G., Mansfield, K.T.: Org. Synth. 49, 1 (1969)CrossRefGoogle Scholar
  27. 27.
    Ullrich, R., Nuske, 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

Copyright information

© Springer International Publishing Switzerland 2016

Authors and Affiliations

  1. 1.PhiladelphiaUSA

Personalised recommendations