Batie C J, Kamin H
J Biol Chem. 1986 Aug 25;261(24):11214-23.
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+.
研究了铁氧化还原蛋白 - NADP⁺还原酶（FNR，或Fd - NADP⁺还原酶）的NADP⁺结合位点的平衡性质，涉及结合特异性以及FNR·NADP⁺复合物的氧化还原性质。除了3'-NADP⁺外，只有2'-腺苷磷酸结合的腺苷核苷酸能与Fd - NADP⁺还原酶结合。解离常数（Kd）值按以下顺序增加：2',5'-ADP＞2',5'-ATP核糖＞NADP⁺＞2'-AMP＞3'-NADP⁺。未发现NAD、NMN或5'-ADP有结合的证据。因此，2'-腺苷磷酸控制底物结合的特异性以及酶活性的特异性。Fd - NADP⁺还原酶对2'-AMP的低亲和力表明，NADP⁺焦磷酸桥的磷酸基团可能也对结合能有显著贡献。发现Fd - NADP⁺还原酶与NADPH形成高亲和力的双电子还原复合物（FNR·NADPH）；复合物的形成与长波长电荷转移带的出现有关。FNR·NADPH复合物的Kd约为氧化型FNR·NADP⁺复合物Kd的6%。正如较低的Kd所预测的，FNR·NADP⁺复合物还原为电荷转移复合物的标准氧化还原电位（Em）比NADP⁺/NADPH电对的电位正约40 mV。快速动力学研究支持将电荷转移复合物描述为主要是氧化型FNR·NADPH。因此，复合物的形成有助于推动电子从黄素蛋白转移到NADP⁺。
