Blue light mediated photoreduction of the flavoprotein NADPH-cytochrome P450 reductase. A Förster-type energy transfer.
The absorption spectra and the corresponding molar absorption coefficients of the fluorophores umbelliferone, FAD and FMN and of the FAD and FMN containing flavoprotein NADPH-cytochrome P450 reductase of different oxydation-reduction states are documented. Binding spectra of the ligand umbelliferone with the CYP2B1:NADPH-cytochrome P450 reductase-complex were determined by difference spectroscopy. The Scatchard plot of the equilibrium ligand binding shows a high affinity part and a low affinity part of 12 and 34 umbelliferone binding sites per CYP2B1:reductase-complex molecule, respectively. The fluorescence excitation and emission spectra of the donor molecule umbelliferone and the acceptor molecules FAD and FMN are given. The fluorescence spectra of the reaction components under test conditions of CYP2B1-dependent 7-ethoxycoumarin-O-deethylase are measured. The excitation energy transfer from the donor umbelliferone (lambda E = 380 nm; lambda F = 460 nm) to the acceptor molecule FMN (lambda E = 465 nm; lambda F = 525 nm) was examined under assay conditions. The results demonstrate that a radiationless Förster-type energy transfer takes place in the presence of the CYP2B1:reductase-complex. It turned out that this effect is a function of the protein complex-concentration. The data presented here combined with previously made observations by Müller-Enoch (Müller-Enoch D. (1994), Z. Naturforsch. 49c, 763-771) support the finding that the umbelliferone molecules, n = 12-34, bound per mole of CYP2B1:reductase-complex, transfer their absorbed light energy radiationless to the FAD binding domain. The complex formed containing 12 or 34 molecules of umbelliferone provides absorption coefficient values at lambda = 380 nm of 78 and 221 mM-1.cm-1, respectively. The Förster-type energy transfer from the donor umbelliferone to the acceptor FAD not only leads to a light activation of the singlet state of FAD but also to a conformational change of the amino acids close to the FAD binding side to favour the encaging of the FAD* triplet state which reacts with the NADPH to form the FADH2 reductase. Due to this process the overall reaction can start with the unquenched excited FAD* triplet state as an intermediate which is about 30 kJ/mol lower in energy than the dark reaction.
Müller-Enoch
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Abteilung Physiologische Chemie, Universität Ulm, Germany.
D000818:Animals; D019362:Cytochrome P-450 CYP2B1 / Q000378:metabolism*; D004735:Energy Transfer; D005486:Flavin Mononucleotide / Q000378:metabolism; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism; D005420:Flavoproteins / Q000737:chemistry / Q000378:metabolism* / Q000528:radiation effects; D008027:Light; D008297:Male; D008862:Microsomes, Liver / Q000187:drug effects / Q000201:enzymology*; D009251:NADPH-Ferrihemoprotein Reductase / Q000737:chemistry / Q000378:metabolism* / Q000528:radiation effects; D010084:Oxidation-Reduction; D010634:Phenobarbital / Q000494:pharmacology; D010777:Photochemistry; D051381:Rats; D017207:Rats, Sprague-Dawley; D013053:Spectrophotometry
D005420:Flavoproteins; D005182:Flavin-Adenine Dinucleotide; D005486:Flavin Mononucleotide; D019362:Cytochrome P-450 CYP2B1; D009251:NADPH-Ferrihemoprotein Reductase; D010634:Phenobarbital