Location of functional centers in the microsomal cytochrome P450 system.
Fluorescence quenching and energy-transfer studies have been carried out to determine the position of FAD and FMN groups of NADPH-cytochrome P450 reductase and of the heme and substrate groups of cytochrome P450 with respect to the lipid/water interphase. Quenching by iodine of the fluorescence of the flavins of the reductase shows a biphasic pattern, due to the different accessibility of FAD and FMN to the solvent with Stern-Volmer constants of 7.9 x 10(-4) and 2.7 x 10(-3) mM-1, respectively. Both prosthetic groups appear to be buried within the three-dimensional structure of the native reductase, FAD more deeply embedded than FMN and with a relative contribution to the total fluorescence of flavins of 84% (FAD) and 16% (FMN). The lack of significant energy transfer (less than 5%) from FAD+FMN to the rhodamine group of the N-labeled phosphatidylethanolamine incorporated in membranes reconstituted with NADPH-cytochrome P450 reductase and phosphatidylcholine points out that both groups are located at a distance greater than 5 nm from the lipid/water interphase. Steady-state fluorescence intensity and anisotropy data obtained with native and FMN-depleted NADPH-cytochrome P450 reductase show that energy transfer between both prosthetic groups occurs in the native reductase with an efficiency of ca. 31%, consistent with a separation between these groups of 2 nm as suggested earlier by Bastiaens, P. I. H., Bonants, P. J. M., Müller, F., & Visser, A. J. W. G. [(1989) Biochemistry 28, 8416-8425] from time-resolved fluorescence anisotropy measurements.(ABSTRACT TRUNCATED AT 250 WORDS)
Centeno
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Departamento de Bioquímica y Biología Molecular, Facultad de Ciencias, Universidad de Extremadura, Badajoz, Spain.
Gutiérrez-Merino
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D000818:Animals; D001665:Binding Sites; D003374:Coumarins / Q000378:metabolism; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry*; D004734:Energy Metabolism; D004735:Energy Transfer; D005486:Flavin Mononucleotide / Q000737:chemistry; D005182:Flavin-Adenine Dinucleotide / Q000737:chemistry; D005453:Fluorescence; D006418:Heme / Q000737:chemistry; D008055:Lipids / Q000737:chemistry; D008862:Microsomes, Liver / Q000737:chemistry*; D009251:NADPH-Ferrihemoprotein Reductase / Q000737:chemistry*; D011487:Protein Conformation; D051381:Rats; D014867:Water / Q000737:chemistry
D003374:Coumarins; D008055:Lipids; D014867:Water; D005182:Flavin-Adenine Dinucleotide; C017299:7-ethoxycoumarin; D006418:Heme; D005486:Flavin Mononucleotide; D003577:Cytochrome P-450 Enzyme System; D009251:NADPH-Ferrihemoprotein Reductase