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


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


cytochrome P450 2B4


cytochrome b5


NADPH cytochrome P450 reductase


4,4′-dithiobis-phenyl azide


potassium phosphate buffer containing 0.25 g/l Emulgen 913


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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

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