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

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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.

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Abbreviations

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

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Authors and Affiliations

  1. Institute of Biomedical Chemistry, Russian Academy of Medical sciences, Pogodinskaya ul. 10, Moscow, Russia

    A. V. Ivanov, A. T. Kopylov, V. G. Zgoda, I. Yu. Toropygin, E. V. Khryapova & Yu. D. Ivanov

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  1. A. V. Ivanov
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  2. A. T. Kopylov
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  3. V. G. Zgoda
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  4. I. Yu. Toropygin
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  5. E. V. Khryapova
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  6. Yu. D. Ivanov
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Correspondence to Yu. D. Ivanov.

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Original Russian Text © A.V. Ivanov, A.T. Kopylov, V.G. Zgoda, I.Yu. Toropygin, E.V. Khryapova, Yu.D. Ivanov, 2009, published in Biomeditsinskaya Khimiya.

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Ivanov, A.V., Kopylov, A.T., Zgoda, V.G. et al. Mass spectrometry identification of cytochrome P450 2B4 interaction sites for NADPH: Cytochrome P450 reductase. Biochem. Moscow Suppl. Ser. B 3, 361–371 (2009). https://doi.org/10.1134/S1990750809040052

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  • DOI: https://doi.org/10.1134/S1990750809040052

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