Advertisement

Mass spectrometry identification of cytochrome P450 2B4 interaction sites for NADPH: Cytochrome P450 reductase

  • A. V. Ivanov
  • A. T. Kopylov
  • V. G. Zgoda
  • I. Yu. Toropygin
  • E. V. Khryapova
  • Yu. D. Ivanov
Experimental Studies

Abstract

The interaction sites for protein partners, cytochrome P450 2B4 (d-2B4) and NADPH: cytochrome P450 reductase (d-Fp), have been identified. These proteins form complexes during their functioning. Nonspecific covalent cross-linking of the d-2B4 complexes with d-Fp in the Emulgen 913 monomerized system was achieved by 4,4′- dithiobis-phenyl azide. Covalently cross-linked peptides of this complex were identified by ESI-MS/MS. Several binding sites have been identified for these proteins. Based on these sites a model for intermolecular interaction between these proteins has been proposed. This model includes 5 contact sites on d-2B4 for d-Fp (stabilized by the cross-linker); these include the following pairs of corresponding peptides of d-2B4 and d-Fp: 1) d-2B4324–336 and d-Fp570–585; 2) d-2B4423–433 and d-Fp102–109; 3) d-2B4327–336 and d Fp452–464; 4) d-2B4192–197 and d-Fp456–464; 5) d-2B4134–139 and d-Fp406–425. In the two last cases d-Fp peptides are located in the interdomain cleft and stabilize the protein-protein complex via the cross-linker and so the d 2B4/d-Fp complex formation by these sites may involve amino acid residues of the peptides d-Fp456–464 and d-Fp406–425, which surround the interdomain cleft.

Key words

cytochrome P450 2B4 NADPH: cytochrome P450 oxidoreductase mass spectrometry dithiobisphenyl azide interaction sites 

Abbreviations used

d-2B4

cytochrome P450 2B4

d-b5

cytochrome b5

d-Fp

NADPH cytochrome P450 reductase

DTBPA

4,4′-dithiobis-phenyl azide

KP/E

potassium phosphate buffer containing 0.25 g/l Emulgen 913

References

  1. 1.
    Porter, T.D. and Coon, M.J., J. Biol. Chem., 1991, vol. 266, pp. 13469–13472.Google Scholar
  2. 2.
    Gotoh, O., J. Biol. Chem., 1992, vol. 267, pp. 83–90.Google Scholar
  3. 3.
    Guengerich, F.P., in Cytochrome P450. Enzyme Systems that Metabolise Drugs and Other Xenobiotics, Ioannides, C. Ed., John Wiley Sons Ltd., 2001.Google Scholar
  4. 4.
    Archakov, A.I. and Bachmanova, G.P., Cytochrome P450 and Active Oxygen, N.Y.: Taylor and Francis, 1990.Google Scholar
  5. 5.
    Imai, Y., J. Biochem., 1981, vol. 89, pp. 351–362.Google Scholar
  6. 6.
    Mayuzumi, H., Shimizu, T., Sambongi, C., Hiroya, K., and Hatano, M., Arch. Biochem. Biophys., 1994, vol. 310, pp. 367–372.CrossRefGoogle Scholar
  7. 7.
    Gruenke, L.D., Konopka, K., Cadieu, M., and Waskell, L., J. Biol. Chem., 1995, vol. 270, pp. 24707–24718.CrossRefGoogle Scholar
  8. 8.
    Vergeres, G. and Waskell, L., Biochimie (Paris), 1995, vol. 77, pp. 604–620.CrossRefGoogle Scholar
  9. 9.
    Wang, M., Roberts, D.L., Paschke, R., Shea, T.M., Masters, B.S.S., and Kim, J.-J.P., Proc. Natl. Acad. Sci. USA., 1997, vol. 94, pp. 8411–8416.CrossRefGoogle Scholar
  10. 10.
    Dean, W.L. and Gray, R.D., J. Biol. Chem., 1982, vol. 257, pp. 14679–14685.Google Scholar
  11. 11.
    Wagner, S.L., Dean, W.L., and Gray, R.D., J. Biol. Chem., 1984, vol. 259, pp. 2390–2395.Google Scholar
  12. 12.
    Lu, A.Y., Levin, W., and Kuntzman, R., Biochem. Biophys. Res. Commun., 1974, vol. 60, pp. 266–272.CrossRefGoogle Scholar
  13. 13.
    Kanaeva, I.P., Dedinskii, I.R., Skotselyas, E.D., Krainev, A.G., Guleva, I.V., Sevryukova, I.F., Koen, Y.M., Kuznetsova, G.P., Bachmanova, G.I., and Archakov, A.I., Arch. Biochem. Biophys., 1992, vol. 298, pp. 395–402.CrossRefGoogle Scholar
  14. 14.
    Kanaeva, I.P., Nikityuk, O.V., Davydov, D.R., Dedinskii, I.R., Koen, Y.M., Kuznetsova, G.P., Scotselyas, E.D., Bachmanova, G.I., and Archakov, A.I., Arch. Biochem. Biophys., 1992, vol. 298, pp. 403–412.CrossRefGoogle Scholar
  15. 15.
    Chang, Y.-T., Stiffelman, O.B., Vakser, I.A., Loew, G.H., Bridges, A., and Waskell, L., Protein Engineering, 1997, vol. 10, pp. 119–129.CrossRefGoogle Scholar
  16. 16.
    Scott, E.E., He, Y.A., Wester, M.R., White, M.A., Chin, C.C., Halpert, J.R., Johnson, E.F., and Stout, C.D., PNAS, 2003, vol. 100, pp. 13196–13201.CrossRefGoogle Scholar
  17. 17.
    Shen, A.L. and Kasper, C.B., J. Biol. Chem., 1995, vol. 270, pp. 27475–27480.CrossRefGoogle Scholar
  18. 18.
    Lehnerer, M., Schulze, J., Achterhold, K., Lewis, D.F., and Hlavica, P., J. Biochem. (Tokyo), 2000, vol. 127, pp. 163–169.Google Scholar
  19. 19.
    Bridges, A., Gruenke, L., Chang, Y.-T., Vakser, I.A., Loew, G., and Waskell, L., J. Biol. Chem., 1998, vol. 273, pp. 17036–17049.CrossRefGoogle Scholar
  20. 20.
    Tamburini, P.P. and Schenkman, J.B., Mol. Pharmacol., 1986, vol. 30, pp. 178–185.Google Scholar
  21. 21.
    Voznesensky, A.I. and Schenkman, J.B., J. Biol. Chem., 1992, vol. 267, pp. 14669–14676.Google Scholar
  22. 22.
    Ivanov, Yu.D., Kanaeva, I.P., Kuznetsov, V.Yu., Lehnerer, M., Schulze, J., Hlavica, P., and Archakov, A.I., Arch. Biochem. Biophys., 1999, vol. 362, pp. 87–93.CrossRefGoogle Scholar
  23. 23.
    Ivanov, Yu.D., Kanaeva, I.P., Gnedenko, O.V., Pozdnev, V.F., Shumyantseva, V.V., Samenkova, N.F., Kuznetsova, G.P., Tereza, A.M., Schmid, R.D., and Archakov, A.I., J. Mol. Recognit., 2001, vol. 14, pp. 185–196.CrossRefGoogle Scholar
  24. 24.
    Muller, E.-C., Lapko, A., Otto, A., Muller, J.J., Ruckpaul, K., and Heinemann, U., Eur. J. Biochem., 2001, vol. 268, pp. 1837–1843.CrossRefGoogle Scholar
  25. 25.
    Gao, Q., Doneanu, C.E., Shaffer, S.A., Adman, E.T., Goodlett, D.R., and Nelson, S.D., J. Biol. Chem., 2006, vol. 281, pp. 20404–20417.CrossRefGoogle Scholar
  26. 26.
    Mikkelsen, R.B. and Wallach, D.F., J. Biol. Chem., 1976, vol. 251, pp. 7413–7416.Google Scholar
  27. 27.
    Dentler, W.L., Pratt, M.M., and Stephens, R.E., J. Cell. Biol., 1980, vol. 84, pp. 381–403.CrossRefGoogle Scholar
  28. 28.
    Zakowski, J.J. and Wagner, R.R., J. Virol., 1980, vol. 36, pp. 93–102.Google Scholar
  29. 29.
    Meisenheimer, K.M. and Koch, T.H., Critical Reviews in Biochem. and Mol. Biol., 1997, vol. 32(2), pp. 101–140.CrossRefGoogle Scholar
  30. 30.
    Karuzina, I.I., Zgoda, V.G., Kuznetsova, G.P., Samenkova, N.F., and Archakov, A.I., Free Radical Biology and Medicine, 1999, vol. 26, pp. 620–632.CrossRefGoogle Scholar
  31. 31.
    Kanaeva, I.P., Skotselyas, E.D., Kuznetsova, G.P., Antonova, G.N., and Bachmanova, G.I., Biokhimiya, 1985, vol. 50, pp. 1382–1388.Google Scholar
  32. 32.
    Spatz, L. and Strittmatter, P., Proc. Natl. Acad. Sci. USA, 1971, vol. 68, pp. 1042–1046.CrossRefGoogle Scholar
  33. 33.
    Laemmli, U.K., Nature, 1970, vol. 227, pp. 680–685.CrossRefGoogle Scholar
  34. 34.
    Shevchenko, A., Keller, P., Scheiffele, P., Mann, M., and Simons, K., Electrophoresis, 1997, vol. 18, pp. 2591–2600.CrossRefGoogle Scholar
  35. 35.
    Kuznetsov, V.Yu., Ivanov, Yu.D., and Archakov, A.I., Proteomics, 2004, vol. 4, pp. 2390–2396CrossRefGoogle Scholar
  36. 36.
    Ivanov, Yu.D., Ivanov, A.V., Petushkova, N.A., Gara, O.G., Kuznetsov, V.Yu., Podoplelov, A.V., and Archakov, A.I., Biomed. Khim. 2008, vol. 54, pp. 435–444.Google Scholar

Copyright information

© Pleiades Publishing, Ltd. 2009

Authors and Affiliations

  • A. V. Ivanov
    • 1
  • A. T. Kopylov
    • 1
  • V. G. Zgoda
    • 1
  • I. Yu. Toropygin
    • 1
  • E. V. Khryapova
    • 1
  • Yu. D. Ivanov
    • 1
  1. 1.Institute of Biomedical ChemistryRussian Academy of Medical sciencesMoscowRussia

Personalised recommendations