A 31P-nuclear-magnetic-resonance study of NADPH-cytochrome-P-450 reductase and of the Azotobacter flavodoxin/ferredoxin-NADP+ reductase complex.
31P-nuclear-magnetic-resonance spectroscopy has been employed to probe the structure of the detergent-solubilized form of liver microsomal NADPH--cytochrome-P-450 reductase. In addition to the resonances due to the FMN and FAD coenzymes, additional phosphorus resonances are observed and are assigned to the tightly bound adenosine 2'-phosphate (2'-AMP) and to phospholipids. The phospholipid content was found to vary with the preparation; however, the 2'-AMP resonance was observed in all preparations tested. In agreement with published results [Otvos et al. (1986) Biochemistry 25, 7220-7228] for the protease-solubilized enzyme, the addition of Mn(II) to the oxidized enzyme did not result in any observable line-broadening of the FMN and FAD phosphorus resonances. The phospholipid resonances, however, were extensively broadened and the line width of the phosphorus resonance assigned to the bound 2'-AMP was broadened by approximately 70 Hz. The data show that only the phosphorus moieties of the phospholipids and the 2'-AMP, but not the flavin coenzymes are exposed to the bulk solvent. Removal of the FMN moiety from the enzyme substantially alters the 31P-NMR spectrum as compared with the native enzyme. The 2'-AMP is removed from the enzyme during the FMN-depletion procedure and the pyrophosphate resonances of the bound FAD are significantly altered. Reconstitution of the FMN-depleted protein with FMN results in the restoration of the coenzyme spectral properties. Reduction of FMN to its air-stable paramagnetic semiquinone form results in broadening of the FMN and 2'-AMP resonances in the detergent-solubilized enzyme. In agreement with previous results. FMN semiquinone formation had little or no effect on the line width of the FMN phosphorus resonance for the proteolytically solubilized enzyme. 31P-NMR experiments with Azotobacter flavodoxin semiquinone, both in its free form and in a complex with spinach ferredoxin-NADP+ reductase, mimic the differential paramagnetic effects of the flavin semiquinone on the line width of the FMN phosphorus resonance, observed by comparison of the detergent-solubilized and protease-solubilized forms of the reductase. The data demonstrate that assignment of the site of flavin semiquinone formation to a particular flavin coenzyme may not always be possible by 31P-NMR experiments in multi-flavin containing enzymes.
Bonants
P J
PJ
Department of Biochemistry, Agricultural University, Wageningen, The Netherlands.
Müller
F
F
Vervoort
J
J
Edmondson
D E
DE
D000818:Animals; D001395:Azotobacter / Q000201:enzymology*; D001665:Binding Sites; D005287:Ferredoxin-NADP Reductase / Q000378:metabolism*; D005486:Flavin Mononucleotide / Q000378:metabolism; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism; D005418:Flavodoxin / Q000378:metabolism*; D005420:Flavoproteins / Q000378:metabolism*; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D008862:Microsomes, Liver / Q000201:enzymology; D009247:NADH, NADPH Oxidoreductases / Q000378:metabolism*; D009251:NADPH-Ferrihemoprotein Reductase / Q000378:metabolism*; D010758:Phosphorus; D011485:Protein Binding; D011817:Rabbits; D013552:Swine
D005418:Flavodoxin; D005420:Flavoproteins; D005182:Flavin-Adenine Dinucleotide; D010758:Phosphorus; D005486:Flavin Mononucleotide; D005287:Ferredoxin-NADP Reductase; D009247:NADH, NADPH Oxidoreductases; D009251:NADPH-Ferrihemoprotein Reductase