Pueyo J J, Gomez-Moreno C, Mayhew S G
Departamento de Bioquímica y Biología Molecular y Celular, Facultad de Ciencias, Universidad de Zaragoza, Spain.
Eur J Biochem. 1991 Dec 18;202(3):1065-71. doi: 10.1111/j.1432-1033.1991.tb16471.x.
The oxidation-reduction potentials of ferredoxin-NADP+ reductase and flavodoxin from the cyanobacterium Anabaena PCC 7119 were determined by potentiometry. The potentials at pH 7 for the oxidized flavodoxin/flavodoxin semiquinone couple (E2) and the flavodoxin semiquinone/hydroquinone couple (E1) were -212 mV and -436 mV, respectively. E1 was independent of pH above about pH 7, but changed by approximately -60 mV/pH below about pH 6, suggesting that the fully reduced protein has a redox-linked pKa at about 6.1, similar to those of certain other flavodoxins. E2 varied by -50 mV/pH in the range pH 5-8. The redox potential for the two-electron reduction of ferredoxin-NADP+ reductase was -344 mV at pH 7 (delta Em = -30 mV/pH). In the 1:1 electrostatic complex of the two proteins titrated at pH 7, E2 was shifted by +8 mV and E1 was shifted by -25 mV; the shift in potential for the reductase was +4 mV. The potentials again shifted following treatment of the electrostatic complex with a carbodiimide, to covalently link the two proteins. By comparison with the separate proteins at pH 7, E2 for flavodoxin shifted by -21 mV and E1 shifted by +20 mV; the reductase potential shifted by +2 mV. The potentials of the proteins in the electrostatic and covalent complexes showed similar pH dependencies to those of the individual proteins. Qualitatively similar changes occurred when ferredoxin-NADP+ reductase from Anabaena variabilis was complexed with flavodoxin from Azotobacter vinelandii. The shifts in redox potential for the complexes were used with previously determined values for the dissociation constant (Kd) of the electrostatic complex of the two oxidised proteins, in order to estimate Kd values for the interaction of the different redox forms of the proteins. The calculations showed that the electrostatic complexes, formed when the proteins differ in their redox states, are stronger than those formed when both proteins are fully oxidized or fully reduced.
通过电位滴定法测定了来自鱼腥藻Anabaena PCC 7119的铁氧化还原蛋白-NADP +还原酶和黄素氧还蛋白的氧化还原电位。在pH 7时，氧化型黄素氧还蛋白/黄素氧还蛋白半醌电对（E2）和黄素氧还蛋白半醌/对苯二酚电对（E1）的电位分别为-212 mV和-436 mV。E1在pH约7以上时与pH无关，但在pH约6以下时每pH变化约-60 mV，这表明完全还原的蛋白质在约6.1处具有氧化还原相关的pKa，与某些其他黄素氧还蛋白类似。E2在pH 5-8范围内每pH变化-50 mV。在pH 7时，铁氧化还原蛋白-NADP +还原酶双电子还原的氧化还原电位为-344 mV（δEm = -30 mV/pH）。在pH 7下滴定的两种蛋白质的1:1静电复合物中，E2偏移了+8 mV，E1偏移了-25 mV；还原酶的电位偏移了+4 mV。在用碳二亚胺处理静电复合物以共价连接两种蛋白质后，电位再次发生偏移。与pH 7时的单独蛋白质相比，黄素氧还蛋白的E2偏移了-21 mV，E1偏移了+20 mV；还原酶电位偏移了+2 mV。静电和共价复合物中蛋白质的电位与单个蛋白质的电位表现出相似的pH依赖性。当可变鱼腥藻的铁氧化还原蛋白-NADP +还原酶与棕色固氮菌的黄素氧还蛋白复合时，发生了定性相似的变化。利用复合物氧化还原电位的偏移以及先前测定的两种氧化蛋白静电复合物的解离常数（Kd）值，来估计蛋白质不同氧化还原形式相互作用的Kd值。计算结果表明，当蛋白质处于不同氧化还原状态时形成的静电复合物，比两种蛋白质都完全氧化或完全还原时形成的静电复合物更强。
