Phosphorus-31 nuclear magnetic resonance and electronic spectroscopic studies of adrenodoxin reductase and its binary...

The 31P NMR spectra of NADPH-adrenodoxin reductase and its complex with NADP+ are reported. The spectrum of adrenodoxin reductase showed two doublets arising from the phosphorus nuclei in the pyrophosphate group of FAD. Both doublets were shifted upfield to different extents in comparison with those of free FAD. Further, one of the doublets of phosphorus nuclei of the pyrophosphate group of bound NADP+ in the complex of adrenodoxin reductase and NADP+ was considerably shifted upfield in comparison with that of free NADP+. The spectrum of the complex of the reductase and NADP+ showed that the resonance of the 2'-phosphate group of NADP+ bound to the reductase was shifted downfield by 1.37 ppm compared with that of free NADP+ in the dianionic state. The 2'-phosphate resonance of bound NADP+ was independent of pH within the physiological range, whereas that of free NADP+ changed according to its ionization. The resonance of the 2'-phosphate group of NADP+ bound to the reductase also revealed that the ratio for the complex of NADP+ and the reductase was 1:1, and that this complex formation was inhibited by a high KCl concentration. These results were confirmed by electronic spectroscopic studies.