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

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Abstract
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 resonance due to the FMN and FAD coenzyme, 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 .apprxeq. 70 Hx. 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.

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