Abstract: Interactions between cytochrome P450 2B4, NADPH:cytochrome P450 reductase and cytochrome b5 have been investigated in the presence of a substrate (7-pentoxyresorufin) and an electron donor, NADPH, in the monomeric reconstituted P450 2B4-contained monooxygenase system. Each partner was immobilized via its amino groups on the carboxymethyldextran biochip surface of an optical biosensor IAsys+. It was shown that, despite immobilization of any of the partners (via their respective amino groups) onto the carboxymethyldextran surface of the IAsys+ optical biosensor, its activity didn't loss. 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, equilibrium dissociation constant (KD) was (0,26±0,13)⋅10-6 M. Comparison of kon, koff and KD for d-Fp/d-2B4 complexes in oxidation conditions with corresponding constants for the oxidized protein forms - (0,10±0,03)⋅106 M-1⋅s-1/ (0,14±0,06) s-1, (0,71±0,37)⋅10-6 M - shows that the decrease in kon and KD occurs due to the increase in lifetime during transition from oxidized to hydroxylation conditions. Complex formation between d-Fp and d-b5 was not registered in oxidation and hydroxylation conditions. The ternary d-Fp/d-2B4/d-b5 complexes formation was shown in hydroxylation and oxidation conditions.
Reference: Ivanov Yu.D., Ivanov A.V., Petushkova N.A., Gara O.G., Kuznetsov V.Yu., Podoplelov A.V., Archakov A.I., The optical biosensor study of the redox partners interactions within the cytochrome P450 2b4 - containing monooxygenase system in hydroxylation conditions, Biomeditsinskaya khimiya, 2008, vol: