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The optical biosensor study of the redox partner interaction with the cytochrome P450 2B4-containing monooxygenase system under hydroxylation conditions

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Biochemistry (Moscow) Supplement Series B: Biomedical Chemistry Aims and scope Submit manuscript

Abstract

The interactions between cytochrome P450 2B4 (d-2B4), NADPH:cytochrome P450 reductase and cytochrome b5 have been investigated in the monomeric reconstituted P450 2B4-containing monooxygenase system in the presence of a substrate (7-pentoxyresorufin) and an electron donor, NADPH. Each partner was immobilized via its amino groups on the carboxymethyldextran biochip surface of the optical biosensor IAsys+. Such mode immobilization was not accompanied by any loss of activities of the immobilized proteins. The formation of binary d-Fp/d-2B4 complexes was registered. The association/dissociation rate constants (kon/koff) were (0.013 ± 0.005) × 106 M−1 s−1/0.05 ± 0.02 s−1, and dissociation constant (KD) was (0.26 ± 0.13) × 10−6 M. Comparison of kon, koff and KD values for d-Fp/d-2B4 complexes formed under hydroxylation (O-dealkylation) with corresponding constants obtained for the oxidized proteins of (0.10 ± 0.03) × 106 M−1 s−1/(0.14 ± 0.06) s−1, and (0.71 ± 0.37) × 10−6 M, respectively shows that the decrease in kon and an insignificant decrease in KD are associated with the increase of complex lifetime during transition from the oxidized to hydroxylation conditions. Complex formation between d-Fp and d-b5 was not registered in both hydroxylation conditions and in the case of oxidized forms of these proteins. In both cases formation of the ternary d-Fp/d-2B4/d-b5 complexes occurred.

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Authors and Affiliations

  1. Orekhovich Institute of Biomedical Chemistry, Russian Academy of Medical Sciences, ul. Pogodinskaya 10, Moscow, 119121, Russia

    Yu. D. Ivanov, A. V. Ivanov, N. A. Petushkova, O. G. Gara, V. Yu. Kuznetsov, A. V. Podoplelov & A. I. Archakov

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  1. Yu. D. Ivanov
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  2. A. V. Ivanov
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  3. N. A. Petushkova
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  4. O. G. Gara
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  5. V. Yu. Kuznetsov
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  6. A. V. Podoplelov
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  7. A. I. Archakov
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Correspondence to Yu. D. Ivanov.

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Original Russian Text © Yu.D. Ivanov, A.V. Ivanov, N.A. Petushkova, O.G. Gara, V.Yu. Kuznetsov, A.V. Podoplelov, A.I. Archakov, 2008, published in Biomeditsinskaya Khimiya.

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Ivanov, Y.D., Ivanov, A.V., Petushkova, N.A. et al. The optical biosensor study of the redox partner interaction with the cytochrome P450 2B4-containing monooxygenase system under hydroxylation conditions. Biochem. Moscow Suppl. Ser. B 2, 367–372 (2008). https://doi.org/10.1134/S1990750808040069

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