Pandini V, Aliverti A, Zanetti G
Dipartimento di Fisiologia e Biochimica Generali, Università degli Studi di Milano, Italy.
Biochemistry. 1999 Aug 17;38(33):10707-13. doi: 10.1021/bi990423h.
Photosystem I of higher plants functions in photosynthesis as a light-driven oxidoreductase producing reduced ferredoxin. Its peripheral subunit PsaD has been identified as the docking site for ferredoxin I. With the aim of elucidating the structure-function relationship and the role of this subunit, a recombinant form of the spinach protein was produced by heterologous expression in Escherichia coli. The PsaD protein was synthesized in soluble form and purified to homogeneity. The interaction of the PsaD subunit with ferredoxin I was investigated using three different approaches: chemical cross-linking between the two purified proteins in solution, affinity chromatography of the PsaD subunit on a ferredoxin-coupled resin, and titration with ferredoxin of the protein fluorescence of the subunit. All these studies indicated that the isolated PsaD in solution has a definite conformation and maintains the ability to bind ferredoxin I with high affinity and specificity. The Kd value of the complex of PsaD and ferredoxin is in the nanomolar range, which is consistent with reported Km values for ferredoxin I photoreduction by thylakoid membranes. The ionic strength dependence of the K(d) suggests that the protein-protein interaction is at least partially electrostatic in nature. Nevertheless, none of the glutamate residues of the acidic cluster of residues 92-94 of ferredoxin I, which have been reported to be involved in the interaction with the subunit, seems to be essential for PsaD binding, as borne out by experiments using ferredoxin I mutants in positions 92-94.
高等植物的光系统I在光合作用中作为一种光驱动氧化还原酶发挥作用，产生还原型铁氧化还原蛋白。其外周亚基PsaD已被确定为铁氧化还原蛋白I的停靠位点。为了阐明该亚基的结构-功能关系及其作用，通过在大肠杆菌中进行异源表达制备了菠菜蛋白的重组形式。PsaD蛋白以可溶形式合成并纯化至同质。使用三种不同方法研究了PsaD亚基与铁氧化还原蛋白I的相互作用：溶液中两种纯化蛋白之间的化学交联、PsaD亚基在铁氧化还原蛋白偶联树脂上的亲和层析以及用铁氧化还原蛋白滴定该亚基的蛋白质荧光。所有这些研究表明，溶液中分离的PsaD具有确定的构象，并保持以高亲和力和特异性结合铁氧化还原蛋白I的能力。PsaD与铁氧化还原蛋白复合物的Kd值在纳摩尔范围内，这与类囊体膜光还原铁氧化还原蛋白I报道的Km值一致。K(d)对离子强度的依赖性表明蛋白质-蛋白质相互作用至少部分是静电性质的。然而，铁氧化还原蛋白I 92-94位酸性簇的谷氨酸残基，据报道参与与该亚基的相互作用，但似乎对PsaD结合并非必不可少，这由使用92-94位铁氧化还原蛋白I突变体的实验证实。
