title: Time-resolved fluorescence spectroscopy of NADPH-cytochrome P-450 reductase: demonstration of energy transfer between the two prosthetic groups.
abstract: Fluorescence as well as fluorescence anisotropy decay parameters have been obtained from NADPH-cytochrome P-450 reductase by time-resolved fluorescence spectroscopy. The two flavins in the enzyme, FMN and FAD, are slightly fluorescent and exhibit heterogeneous fluorescence lifetimes, as observed with other flavoproteins. The time-dependent anisotropy is also multiexponential and is wavelength-dependent. The anisotropy decay is biexponential with two correlation times when the enzyme is excited at the red edge of the first absorption band (514 nm). When the enzyme is excited in the light absorption maximum (458 nm), an additional shorter correlation time is found, which contains information about the rate of energy transfer between the two flavins present in the enzyme. FMN-depleted NADPH-cytochrome P-450 reductase shows also only two correlation times, as does the enzyme in the "air-stable" semiquinone state when excited at 458 nm. Wavelength-dependent steady-state anisotropy measurements of native and FMN-depleted protein show that the former exhibits lower values than the latter in the region of the first absorption band, but when the red edge of the absorption band is reached, the anisotropy becomes equal in both preparations. A similar situation is encountered in model compounds, monomeric and dimeric flavins, immobilized in poly(methyl methacrylate). Both in the models and in the flavoprotein this can be attributed to failure of energy transfer at the red edge of the absorption band. From the results we were able to derive both geometric parameters and dynamic properties of both flavins in the NADPH-cytochrome P-450 reductase.(ABSTRACT TRUNCATED AT 250 WORDS)
authors.affiliation: Department of Biochemistry, Agricultural University, Wageningen, The Netherlands.
authors.forename: P I
authors.initials: PI
authors.lastname: Bastiaens
authors.forename: P J
authors.initials: PJ
authors.lastname: Bonants
authors.forename: F
authors.initials: F
authors.lastname: Müller
authors.forename: A J
authors.initials: AJ
authors.lastname: Visser
mesh_terms: D055598:Chemical Phenomena; D002627:Chemistry, Physical; D004735:Energy Transfer; D005486:Flavin Mononucleotide; D005182:Flavin-Adenine Dinucleotide; D005454:Fluorescence Polarization; D008433:Mathematics; D009251:NADPH-Ferrihemoprotein Reductase; D013050:Spectrometry, Fluorescence; D013997:Time Factors
chemical_list: D005182:Flavin-Adenine Dinucleotide; D005486:Flavin Mononucleotide; D009251:NADPH-Ferrihemoprotein Reductase