An essential carboxyl group at the nucleotide binding site of ferredoxin-NADP+ oxidoreductase.
Woodward's reagent K (N-ethyl-5-phenylisoxazolium-3'-sulfonate) inactivated both soluble and membrane bound-ferredoxin-NADP+ reductase of spinach chloroplasts. Either NADP+ or NADPh afforded complete protection against modification. Ki and the apparent Kd for protection afforded by NADP+ depended on the ionic strength of the medium. Nucleophylic displacement of reagent bound to the soluble enzyme by [14C]glycine ethyl ester showed that 5 to 6 carboxyl groups/flavin were modified when the diaphorase activity was completely inhibited. In differential labeling experiments using NADP+ as protective agent, it was shown that enzyme inactivation was due to blocking of only 1 carboxyl group/mol. Derivatized reductase did not bind pyridine nucleotides. Protection by NADP+ of the membrane-bound reductase was higher, and the apparent Kd for NADP+ lower, in the light than in the dark. Inactivation increased abruptly with the external pH, indicating a progressive exposure of the carboxyl group as the pH was raised. The results presented suggest (a) the existence of a light-driven conformational change and a pH-dependent transition in membrane-bound ferredoxin-NADP+ reductase; (b) the presence of an essential carboxyl residue in the nucleotide binding site of the reductase.
D001665:Binding Sites; D002736:Chloroplasts / Q000201:enzymology*; D008058:Dihydrolipoamide Dehydrogenase / Q000378:metabolism; D005287:Ferredoxin-NADP Reductase / Q000378:metabolism*; D007700:Kinetics; D008433:Mathematics; D009247:NADH, NADPH Oxidoreductases / Q000378:metabolism*; D009249:NADP; D010084:Oxidation-Reduction; D010944:Plants / Q000201:enzymology; D011485:Protein Binding; D013053:Spectrophotometry
D009249:NADP; D005287:Ferredoxin-NADP Reductase; D009247:NADH, NADPH Oxidoreductases; D008058:Dihydrolipoamide Dehydrogenase