Product Autofluorescence Activation in the Cytochrome P450 Monooxygenase System

Abstract

The relative increase of the 7-ethoxycoumarin-O-deethylase and the 6,7-dimethoxycoumarin-O-demethylase activities were found 93 and 236% using a reconstituted cytochrome P4502B1:NADPH-P450 reductase system by adding to the reaction mixtures their own products. The assays were irradiated during the reactions with the excitation wavelength maximum of their products umbelliferone (lambda E = 365 nm) or scopoletin (lambda E 98 nm), respectively. Addition of the products to the reaction mixtures without irradiation (dark reaction) had no activating effect on the specific activities of the 7-ethoxycoumarin-O-deethylase or the 6,7-dimethoxycoumarin-O-demethylase. The relative increase of the specific activities is dependent on the excitation light intensities and was at maximum when the light intensity at the sample cuvette was 0.4 mW/cm2. The activation energies of the P4502B1-dependent 7-ethoxycoumarin-O-deethylation reaction obtained from Arrhenius plots with and without added umbelliferone and irradiation with lambda E = 365 nm are 14.7 kJ/mol and 33.5 kJ/mol, respectively, in the temperature range of 27-37 degrees C. The irradiation energy of the fluorescent product umbelliferone change the catalytic mechanism, which has a two times lower activation energy in the presence of the irradiated product umbelliferone. Umbelliferone and scopoletin have highest fluorescence intensities in the wavelength range of the blue light (440-480 nm). The photochemical action spectrum of the 7-ethoxycoumarin-O-deethylase of the P4502B1:reductase system is also found to be in the wavelength range of 420-470 nm. No activation effect was seen with irradiating light lower than 400 nm.(ABSTRACT TRUNCATED AT 250 WORDS)