Complex-forming properties of spinach NADP+ reductase with ferredoxin, ferrocyanide and NADP+.
The flavoprotein NADP+ reductase from spinach chloroplasts may form a ternary complex with one molecule of NADP+ and one molecule of ferredoxin. Spectroscopic titration studies show that the NADP+ binding site and the ferredoxin binding site are totally independent, that is previous binding of ferredoxin does not modify binding of NADP+, and conversely. Since NADP+ reductase conditions the diaphorase reaction, that is an electron transfer between NADPH and various acceptors such as ferricyanide, the binding of ferrocyanide and its possible interaction with NADP+ and ferredoxin has been studied. Ferrocyanide behaves as a competitive inhibitor with respect to both NADP+ and ferredoxin. This seems paradoxical since NADP+ and ferredoxin are independently bound at two different non-overlapping sites of the flavoprotein. This apparent paradox may be resolved by a theoretical analysis of the interactions between either ferrocyanide and NADP+, or ferrocyanide and ferredoxin. Theory shows that if ferrocyanide is non-specifically bound at two independent sites, namely the NADP+ and the ferredoxin binding sites, it appears competitive with respect to both NADP+ and ferredoxin, although ternary flavoprotein-ferredoxin-ferrocyanide and flavoprotein-NADP+-ferrocyanide complexes are formed. The binding constants of NADP+, ferredoxin and ferrocyanide for the enzyme have been determined. These results are discussed in connection with the possible mechanism of the diaphorase reaction.
D000494:Allosteric Regulation; D001665:Binding Sites; D005288:Ferredoxins / Q000378:metabolism*; D005295:Ferrocyanides / Q000378:metabolism*; D007700:Kinetics; D008024:Ligands; D009247:NADH, NADPH Oxidoreductases / Q000378:metabolism*; D009249:NADP / Q000378:metabolism*; D009252:NADPH Dehydrogenase / Q000378:metabolism*; D010944:Plants; D011485:Protein Binding; D011487:Protein Conformation; D013057:Spectrum Analysis
D005288:Ferredoxins; D005295:Ferrocyanides; D008024:Ligands; D009249:NADP; D009247:NADH, NADPH Oxidoreductases; D009252:NADPH Dehydrogenase