Association of ferredoxin-NADP+ reductase with NADP(H) specificity and oxidation-reduction properties.
The equilibrium properties of the NADP+ binding site of ferredoxin-NADP+ reductase (FNR, or Fd-NADP+ reductase) were examined with regard to specificity in binding, and with regard to the oxidation-reduction properties of the FNR.NADP+ complex. With the exception of 3'-NADP+, only adenosine nucleotides with a 2'-adenosyl phosphate bound to Fd-NADP+ reductase. Kd values increased in the order: 2',5'-ADP greater than 2',5'-ATP ribose greater than NADP+ greater than 2'-AMP greater than 3'-NADP+. No evidence was found for binding of NAD, NMN, or 5'-ADP. Thus the 2'-adenosylphosphate controls specificity in substrate binding, as well as specificity in enzyme activity. The low affinity of Fd-NADP+ reductase for 2'-AMP suggests that the phosphate(s) of the pyrophosphate bridge of NADP+ may also contribute significantly to binding energy. Fd-NADP+ reductase was found to form a high-affinity two-electron reduced complex (FNR.NADPH) with a NADPH; complex formation was associated with appearance of long-wavelength charge-transfer bands. Kd of FNR.NADPH complex was about 6% the Kd of oxidized FNR.NADP+ complex. As predicted by the lower Kd, the Em for reduction of FNR.NADP+ complex to the charge-transfer complex was about 40 mV more positive than the potential of the NADP+/NADPH couple. Rapid kinetic studies supported description of the charge-transfer complex as primarily oxidized FNR.NADPH. Thus, complex formation helps drive electron transfer from the flavoprotein to NADP+.
D000227:Adenine Nucleotides / Q000378:metabolism; D001665:Binding Sites; D008058:Dihydrolipoamide Dehydrogenase / Q000037:antagonists & inhibitors; D005287:Ferredoxin-NADP Reductase / Q000378:metabolism*; D007700:Kinetics; D009247:NADH, NADPH Oxidoreductases / Q000378:metabolism*; D009249:NADP / Q000378:metabolism*; D010084:Oxidation-Reduction; D011199:Potentiometry; D013379:Substrate Specificity
D000227:Adenine Nucleotides; D009249:NADP; D005287:Ferredoxin-NADP Reductase; D009247:NADH, NADPH Oxidoreductases; D008058:Dihydrolipoamide Dehydrogenase