Localization of the free radical on the flavin mononucleotide of the air-stable semiquinone state of NADPH-cytochrome P-450 reductase using 31P NMR spectroscopy.
Microsomal NADPH-cytochrome P-450 reductase is the only mammalian flavoprotein known to contain both FAD and FMN as prosthetic groups. The discovery of the air-stable semiquinone [Masters, B. S. S., Kamin, H., Gibson, Q. H., & Williams, C. H., Jr. (1965) J. Biol. Chem. 240, 921-931] and its identification as a one-electron-reduced state [Iyanagi, T., & Mason, H. S. (1973) Biochemistry 12, 2297-2308] have engendered a number of studies to elucidate its unique catalytic mechanism. In this paper, 31P NMR spectroscopy is utilized to probe the localization of the free radical in this air-stable semiquinone form and to ascertain the environments of the FAD and FMN prosthetic groups as affected by the paramagnetic ion Mn(II). Consistent with conclusions drawn from studies utilizing FMN-free reductase [Vermilion, J. L., & Coon, M. J. (1978) J. Biol. Chem. 253, 8812-8819], the free radical was shown to reside on the FMN moiety by the broadening of its characteristic resonance in the 31P NMR spectrum. In addition, the effect of the paramagnetic ion Mn(II) was determined on the four resonances attributable to FAD and FMN and the additional ones contributed by NADP+ resulting from the oxidation of the physiological reductant NADPH. The addition of Mn(II) had little effect on the line widths of the FMN and FAD signals but resulted in an increase in their intensities due to a decrease in T1 relaxation times.(ABSTRACT TRUNCATED AT 250 WORDS)
D000818:Animals; D004795:Enzyme Stability; D005486:Flavin Mononucleotide / Q000378:metabolism*; D005609:Free Radicals; D008099:Liver / Q000201:enzymology*; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D008345:Manganese / Q000494:pharmacology; D009249:NADP / Q000378:metabolism; D009251:NADPH-Ferrihemoprotein Reductase / Q000302:isolation & purification / Q000378:metabolism*; D010758:Phosphorus; D011485:Protein Binding; D011809:Quinones; D013552:Swine
D005609:Free Radicals; D011809:Quinones; D010758:Phosphorus; D008345:Manganese; D009249:NADP; D005486:Flavin Mononucleotide; D009251:NADPH-Ferrihemoprotein Reductase