A 31 P‐nuclear‐magnetic‐resonance study of NADPH–cytochrome‐ P ‐450 reductase and of the Azotobacter flavodoxin/ferredoxin‐NADP + reductase complex

31 P‐nuclear‐magnetic‐resonance spectroscopy has been employed to probe the structure of the detergentsolubilized 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 ∼ 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 31 P‐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. 31 P‐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 31 P‐NMR experiments in multi‐flavin containing enzymes.