Riedel K U, Jouanneau Y, Masepohl B, Pühler A, Klipp W
Lehrstuhl für Genetik, Fakultät für Biologie, Universität Bielefeld, Germany.
Eur J Biochem. 1995 Aug 1;231(3):742-6. doi: 10.1111/j.1432-1033.1995.tb20756.x.
The fdxN gene from Rhizobium meliloti encoding a bacterial-type ferredoxin (FdxN) was expressed in Escherichia coli under the control of the lac promoter. The fdxN gene product was purified under anaerobic conditions by ion-exchange chromatography and gel-filtration steps using an antiserum raised against an FdxN-LacZ fusion protein as a detection system. The purified ferredoxin was shown to be identical to the predicted R. meliloti FdxN protein in its amino acid composition and N-terminal amino acid sequence. Chemical determination of the iron content revealed 8.6 +/- 0.6 mol Fe/mol FdxN. The ultraviolet/visible absorption spectrum of the FdxN protein in the oxidized form exhibited maxima at 284 nm and 378 nm, with an A378/A284 ratio of 0.7. EPR spectroscopy revealed a rhombic signal when FdxN was partially reduced, and a broad signal indicative of spin-spin interaction when fully reduced, suggesting the presence of two Fe-S cluster/ferredoxin polypeptide. Our data suggest that FdxN contains two [4Fe-4S] clusters. Purified FdxN was able to mediate electron transport between illuminated chloroplasts and Rhodobacter capsulatus nitrogenase in vitro.
来自苜蓿根瘤菌的编码细菌型铁氧化还原蛋白（FdxN）的fdxN基因在乳糖启动子的控制下于大肠杆菌中表达。利用针对FdxN-LacZ融合蛋白产生的抗血清作为检测系统，通过离子交换色谱和凝胶过滤步骤在厌氧条件下纯化fdxN基因产物。纯化后的铁氧化还原蛋白在氨基酸组成和N端氨基酸序列方面与预测的苜蓿根瘤菌FdxN蛋白相同。铁含量的化学测定显示，每摩尔FdxN含有8.6±0.6摩尔铁。氧化形式的FdxN蛋白的紫外/可见吸收光谱在284nm和378nm处有最大值，A378/A284比值为0.7。电子顺磁共振光谱显示，当FdxN部分还原时出现菱形信号，完全还原时出现表明自旋-自旋相互作用的宽信号，这表明存在两个铁硫簇/铁氧化还原蛋白多肽。我们的数据表明FdxN含有两个[4Fe-4S]簇。纯化后的FdxN能够在体外介导光照下的叶绿体与荚膜红细菌固氮酶之间的电子传递。
