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

Abstract

'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 -72281 for the protease-solubilized enzyme, the addition of Mn(I1) 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 z 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. ferredoxin-NADP' reductase (EC 1.18.1.2). P-450. During this electron transfer, the FAD and FMN coenzymes function at both semiquinone and hydroquinone redox levels.31P-NMR spectroscopy is a powerful technique to investigate the environments of phosphorus groups in proteins and has been profitably used to study the flavin coenzyme and protein-bound phosphorus residues in Megasphaera elsdenii flavodoxin [18], Aspergillus niger glucose oxidase [19], bovine milk xanthine oxidase [20], and Azotobucter vinelandii flavodoxin [21, 221. Otvos et al. [23] have recently investigated the properties of the bound flavin phosphates in the proteolytically solubilized form of the P-450 reductase, by 31P NMR. The work reported here, on the detergent-solubilized form of the reductase, represents an extension of their published studies.In addition, it is of importance to compare the properties of the two forms in as much as the detergent-solubilized enzyme retains activity in the cytochrome P-450 assay while the proteolytically solubilized form does not [16, 171. As documented in this paper, the most significant difference in the 31P-NMR spectral properties of the two forms of the enzyme is that the FMN phosphorus resonance is extensively broadened on formation of the air-stable FMN

Three-dimensional structure of NADPH–cytochrome P450 reductase: Prototype for FMN- and FAD-containing enzymes

“…This distinct domain arrangement is consistent with results from further studies in which each domain was expressed independently (14), and unfolding of the polypeptide was correlated with the release of each individual flavin (15). CPR has been studied extensively by a number of biophysical methods, including EPR spectroscopy (16,17), time-resolved fluorescence spectroscopy (18,19), 31 P NMR (20,21), and resonance Raman spectroscopy (22). Despite these intensive studies, little has been determined about physical and electronic interactions between FMN, FAD, and NADPH and interactions between CPR and its electron transfer partners.…”

Crystallization and preliminary x-ray studies of NADPH-cytochrome P450 reductase.

“…EPR spectroscopy (29,30), timeresolved fluorescence spectroscopy (31, 32), 31P NMR spectroscopy (10,(33)(34)(35)(36), and resonance Raman spectroscopy (37,38) *To whom reprint requests should be addressed.…”

31P Nuclear magnetic resonance study of the flavoprotein component of the Escherichia coli sulfite reductase

“…In contrast, the absence of any effect of manganese is interpreted as an indication that the phosphorus nucleus is buried deeply enough in the protein interior to preclude the influence of the paramagnetic ion. In addition, the observation of an increase in intensity as a direct consequence of more efficient spin‐lattice relaxation, with each resonance being less attenuated by the effects of incomplete relaxation between pulses has been observed upon addition of Mn(II) for cytochrome P450 reductase [17–19]. This observation indicated that the phosphate‐containing regions of protein‐bound FMN and FAD were shielded from interaction with bulk solvent, but were still sensitive to the influence of paramagnetic ions.…”

“…The three main phosphorus resonances observed were easily attributed to the phosphate groups of bound FMN and bound FAD from their chemical shifts and by comparison with 31 P‐NMR spectra obtained with cytochrome P450 reductase [17–19]. However, the exact value of the different chemical shifts differs from those observed in cytochrome P450 reductase suggesting that the environment of the flavin phosphate groups, in SiR‐FP, are somewhat different from that in cytochrome P450 reductase.…”

“…31 P‐NMR spectroscopy has already been employed with success for studying a number of proteins containing flavin or phosphorus [14–19], including SiR‐FP related proteins such as cytochrome P450 reductase [17–19] or flavodoxins [16]. For the first time, it was used here to study SiR‐FP60 structural properties.…”