Lambeth J D, McCaslin D R, Kamin H
J Biol Chem. 1976 Dec 10;251(23):7545-50.
Adrenodoxin reductase and adrenodoxin have been shown (Chu, J.-W., and Kimura, T. (1973) J. Biol. Chem. 248, 5183-5187) to form a low dissociation constant, 1:1 complex when both proteins are in the oxidized form. We have found that when adrenodoxin: adrenodoxin reductase ratios are varied by increasing the adrenodoxin concentration, with adrenodoxin reductase held constant, an increasing rate of cytochrome c reduction, with NADPH as reductant, is seen up to a ratio of 1:1, indicating that cytochrome c reduction occurs via the protein-protein complex. Spectra observed during titration of this protein-protein complex with NADH were resolved into components by the linear programming method, using a computer program written in Fortran IV. Analysis of the data has shown that the flavoprotein is reduced prior to the iron sulfur protein, and that the midpoint oxidation-reduction potentials (pH 7.5) of the two proteins are -295 and -331 mV, respectively, when both are present in the complex. Complex formation does not alter the potential of adrenodoxin reductase, but changes that of adrenodoxin by -40 mV. Equilibrium constants derived from potential measurements show that the strength of the protein-protein interaction in the complex is unaltered by reduction of adrenodoxin reductase, but is decreased by about 1 kcal due to reduction of adrenodoxin. The low dissociation constants for both oxidized reduced forms of the adrenodoxin reductase-adrenodoxin complex indicate that the complex must remain associated throughout its catalytic cycle. Titration of the adrenodoxin reductase-adrenodoxin complex with the physiologic reductant, NADPH, was followed by EPR and visible spectra, and yielded an order of reduction of the components identical with that seen when NADH was used as reductant. Reduction of the protein-protein complex with NADPH yielded a ternary complex between NADP+, flavoprotein, and iron sulfur protein, with the two electrons located in a "charge transfer" complex between flavoprotein and pyridine nucleotide.
肾上腺皮质铁氧化还原蛋白还原酶和肾上腺皮质铁氧化还原蛋白已被证明（朱，J.-W.，和木村，T.（1973年）《生物化学杂志》248，5183 - 5187），当两种蛋白质都处于氧化形式时，会形成一种低解离常数的1:1复合物。我们发现，当通过增加肾上腺皮质铁氧化还原蛋白的浓度来改变肾上腺皮质铁氧化还原蛋白与肾上腺皮质铁氧化还原蛋白还原酶的比例，而肾上腺皮质铁氧化还原蛋白还原酶保持恒定时，以NADPH作为还原剂，细胞色素c的还原速率会不断增加，直至达到1:1的比例，这表明细胞色素c的还原是通过蛋白质 - 蛋白质复合物发生的。在用NADH滴定这种蛋白质 - 蛋白质复合物过程中观察到的光谱，通过使用用Fortran IV编写的计算机程序，采用线性规划方法分解为各个组分。数据分析表明，黄素蛋白在铁硫蛋白之前被还原，并且当两种蛋白质都存在于复合物中时，它们的中点氧化还原电位（pH 7.5）分别为 - 295和 - 331 mV。复合物的形成不会改变肾上腺皮质铁氧化还原蛋白还原酶的电位，但会使肾上腺皮质铁氧化还原蛋白的电位改变 - 40 mV。从电位测量得出的平衡常数表明，复合物中蛋白质 - 蛋白质相互作用的强度不会因肾上腺皮质铁氧化还原蛋白还原酶的还原而改变，但由于肾上腺皮质铁氧化还原蛋白的还原会降低约1千卡。肾上腺皮质铁氧化还原蛋白还原酶 - 肾上腺皮质铁氧化还原蛋白复合物的氧化还原形式的低解离常数表明，该复合物在其整个催化循环中必须保持结合状态。用生理还原剂NADPH滴定肾上腺皮质铁氧化还原蛋白还原酶 - 肾上腺皮质铁氧化还原蛋白复合物，随后进行EPR和可见光谱分析，得到的组分还原顺序与使用NADH作为还原剂时相同。用NADPH还原蛋白质 - 蛋白质复合物会产生一种NADP⁺、黄素蛋白和铁硫蛋白之间的三元复合物，两个电子位于黄素蛋白和吡啶核苷酸之间的“电荷转移”复合物中。
