Zanetti G, Aliverti A, Curti B
J Biol Chem. 1984 May 25;259(10):6153-7.
The water-soluble carbodiimide, N-ethyl-3-(3-dimethylaminopropyl)carbodiimide was found to effectively cross-link ferredoxin to ferredoxin-NADP+ reductase. The covalent complex has a stoichiometry of 1 mol of ferredoxin per mol of the reductase. The flavoprotein moiety of the cross-linked complex maintains most of its diaphorase activity and more interestingly has gained the capacity to catalyze the NADPH-cytochrome c reaction without addition of free ferredoxin in the assay mixture. Furthermore, the cross-linked complex binds NADP+ with a Kd = 88 microM at an ionic strength of 0.02 M. These results show that a ternary complex among the reductase and its substrates can be formed, suggesting that the binding sites for ferredoxin and the pyridine nucleotides are distinct. The bound ferredoxin can interact with cytochrome c; the iron-sulfur cluster of the cross-linked complex is shown to be reduced under anaerobic conditions by NADPH and to be required for the catalysis of the NADPH-cytochrome c reductase reaction. The cross-linked complex, added to thylakoids inhibited by the antibody against the reductase, catalyzes the H2O-cytochrome c photoreduction, which suggests that the ferredoxin moiety of the complex can interact with its electron donor in the photosynthetic chain. Restoration of NADP+ photoreduction requires the addition of free ferredoxin.
发现水溶性碳二亚胺N-乙基-3-(3-二甲基氨基丙基)碳二亚胺能有效地将铁氧化还原蛋白与铁氧化还原蛋白-NADP⁺还原酶交联。共价复合物的化学计量比为每摩尔还原酶含1摩尔铁氧化还原蛋白。交联复合物的黄素蛋白部分保持了其大部分的递氢酶活性，更有趣的是，在测定混合物中不添加游离铁氧化还原蛋白的情况下，它获得了催化NADPH-细胞色素c反应的能力。此外，在离子强度为0.02 M时，交联复合物以Kd = 88 μM的亲和力结合NADP⁺。这些结果表明，还原酶与其底物之间可以形成三元复合物，这表明铁氧化还原蛋白和吡啶核苷酸的结合位点是不同的。结合的铁氧化还原蛋白可以与细胞色素c相互作用；交联复合物的铁硫簇在厌氧条件下被NADPH还原，并且是催化NADPH-细胞色素c还原酶反应所必需的。将交联复合物添加到被抗还原酶抗体抑制的类囊体中，能催化H₂O-细胞色素c光还原，这表明复合物中的铁氧化还原蛋白部分可以与其在光合链中的电子供体相互作用。恢复NADP⁺光还原需要添加游离铁氧化还原蛋白。
