On the ligand-protein and ligand-flavin interactions in NADPH-adrenodoxin reductase as studied by 31P- and 13C-NMR. Use of 13C-enriched FAD as a probe.
The interaction between 2',5'-ADP and NADPH-adrenodoxin reductase from bovine adrenocortical mitochondria was examined by titrating the enzyme with 2',5'-ADP, while the 31P-signals of 2',5'-ADP were being monitored by 31P-NMR. From the titration profile, the dissociation constant for the complex of the enzyme with 2',5'-ADP was estimated to be 0.22 +/- 0.05 mM. Adrenodoxin reductase was reconstituted with 13C-enriched FADs. The 13C-enriched FADs used were [2-13C]-, [4,10 alpha-13C2]-, and [4 alpha-13C]FAD. The 13C-NMR spectra of these reconstituted enzyme preparations showed 13C-resonance peaks corresponding to the enriched carbon atoms at 160.6 , 165.1, 136.6, and 152.4 ppm (2-, 4-, 4 alpha-, and 10 alpha-13C atoms, respectively). When 2',5'-ADP was bound to the reconstituted enzyme, these 13C-resonance peaks did not shift appreciably from those of the unbound enzyme, whereas in the complex of the reconstituted enzyme with NADP+, the signals for 4- and 10 alpha-13C shifted to higher fields by 2.1 and 0.7 ppm, respectively and the 4 alpha-13C signal shifted to a lower field by 1.4 ppm. These results suggest that in the complex of the enzyme with NADP+ the pyridine moiety is located in the vicinity of C(4 alpha)-C(4) region and that the pi-electron density of the 4 alpha-position of flavin is decreased in the enzyme-NADP+ complex. This argues in favor of the electron transfer from the dihydropyridine moiety of NADPH to the electron-deficient N(5) = C(4 alpha) region of flavin.
Fujii
S
S
Laboratory of Chemistry, Kansai Medical University, Osaka.
Nonaka
Y
Y
Okamoto
M
M
Miura
R
R
D000244:Adenosine Diphosphate / Q000378:metabolism; D000302:Adrenal Cortex / Q000201:enzymology; D000818:Animals; D001665:Binding Sites; D002417:Cattle; D005287:Ferredoxin-NADP Reductase / Q000737:chemistry / Q000378:metabolism*; D005182:Flavin-Adenine Dinucleotide; D066298:In Vitro Techniques; D009682:Magnetic Resonance Spectroscopy; D015335:Molecular Probes
D015335:Molecular Probes; D005182:Flavin-Adenine Dinucleotide; C045716:adenosine 2',5'-diphosphate; D000244:Adenosine Diphosphate; D005287:Ferredoxin-NADP Reductase