Batie C J, Kamin H
J Biol Chem. 1984 Jul 25;259(14):8832-9.
Ferredoxin:NADP+ oxidoreductase (ferredoxin: NADP+ reductase, EC 1.18.1.2) was shown to form a ternary complex with its substrates ferredoxin (Fd) and NADP(H), but the ternary complex was less stable than the separate binary complexes. Kd for oxidized binary Fd-ferredoxin NADP+ reductase complex was less than 50 nM; Kd(Fd) increased with NADP+ concentration, approaching 0.5-0.6 microM when the flavoprotein was saturated with NADP+ K(NADP+) also increased from about 14 microM to about 310 microM, on addition of excess Fd. The changes in Kd were consistent with negative cooperativity between the associations of Fd and NADP+ and with our unpublished observations which suggest that product dissociation is rate-limiting in the reaction mechanism. Similar interference in binding was observed in more reduced states; NADPH released much ferredoxin:NADP+ reductase from Fd-Sepharose whether the proteins were initially oxidized or reduced. Complexation between Fd and ferredoxin: NADP+ reductase was found to shield each center from paramagnetic probes; charge specificity suggested that the active sites of Fd and ferredoxin:NADP+ reductase were, respectively, negatively and positively charged.
NADP⁺氧化还原酶（铁氧化还原蛋白：NADP⁺还原酶，EC 1.18.1.2）已被证明能与其底物铁氧化还原蛋白（Fd）和NADP(H)形成三元复合物，但该三元复合物比单独的二元复合物稳定性更低。氧化态二元Fd - 铁氧化还原蛋白NADP⁺还原酶复合物的解离常数（Kd）小于50 nM；Kd(Fd)随NADP⁺浓度增加而增大，当黄素蛋白被NADP⁺饱和时接近0.5 - 0.6 μM。加入过量Fd后，K(NADP⁺)也从约14 μM增加到约310 μM。Kd的变化与Fd和NADP⁺结合之间的负协同性一致，也与我们未发表的观察结果相符，即产物解离在反应机制中是限速步骤。在更多还原态下也观察到类似的结合干扰；无论蛋白质最初是氧化态还是还原态，NADPH都会从Fd - 琼脂糖中释放出大量铁氧化还原蛋白：NADP⁺还原酶。发现Fd与铁氧化还原蛋白：NADP⁺还原酶之间的复合作用能使每个中心免受顺磁探针的影响；电荷特异性表明Fd和铁氧化还原蛋白：NADP⁺还原酶的活性位点分别带负电荷和正电荷。
