Characterization of BphF, a Rieske-type ferredoxin with a low reduction potential
Characterization of BphF, a Rieske-type ferredoxin with a low reduction potential
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DOI：
10.1021/bi001780r
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发表时间：
2001-01-09
期刊：
影响因子：
2.9
通讯作者：
Eltis, LD
中科院分区：
文献类型：
--
作者：
Couture, MMJ;Colbert, CL;Eltis, LD
BphF是一个小的，可溶的，rieske型铁氧还蛋白参与微生物降解联苯。对异种表达的、带有his标记的BphF进行快速厌氧纯化，每升细胞培养物产生15毫克高度均匀的重组蛋白rcBphF。采用高取向热解石墨（HOPG）电极测定rcBphF的还原电位为-157 +/- 2 mV，相对于标准氢电极（SHE） （20 mM MOPS, 80 mM KCI， 1 nM二硫苏糖醇，pH 7.0, 22℃）。还原后的rcBphF的电子顺磁共振谱为典型的Rieske簇，而rcBphF的圆二色性（CD）谱与苯双加氧酶系统的同源蛋白BedB的相似，表明这两种蛋白的簇环境高度保守。rcBphF的还原电位和CD谱与pH值在5 ~ 10之间相对独立，表明其组氨酸基配体的kp (2)s不在此范围内，凝胶过滤研究表明rcBphF在溶液中容易低聚。以甲酸钠或聚乙二醇（PEG）为主要沉淀剂，制备了rcBphF晶体。对晶体分子间接触的分析揭示了一种头对尾的相互作用，这种相互作用遮挡了簇，但在溶液中不太可能发现。rcBphF在溶液中的寡聚化通过添加二硫苏糖醇而被逆转，并且与非共价晶体相互作用无关。此外，rcBphF的寡聚化状态不影响后者的还原电位。这些结果表明，与双氧酶相关的铁氧还毒素和线粒体bc(1)复合物的Rieske簇的还原电位450 mV的扩散不是由于它们的溶剂暴露的显著差异。
BphF is a small, soluble, Rieske-type ferredoxin involved in the microbial degradation of biphenyl. The rapid, anaerobic purification of a heterologously expressed, his-tagged BphF yielded 15 mg of highly homogeneous recombinant protein, rcBphF, per liter of cell culture. The reduction potential of rcBphF, determined using a highly oriented pyrolytic graphite (HOPG) electrode, was -157 +/- 2 mV vs the standard hydrogen electrode (SHE) (20 mM MOPS, 80 mM KCI, and 1 nM dithiothreitol, pH 7.0, 22 degreesC). The electron paramagnetic resonance spectrum of the reduced rcBphF is typical of a Rieske cluster while the close similarity of the circular dichroic (CD) spectra of rcBphF and BedB, a homologous protein from the benzene dioxygenase system, indicates that the environment of the cluster is highly conserved in these two proteins. The reduction potential and CD spectra of rcBphF were relatively independent of pH between 5 and 10, indicating that the p K (2)s of the cluster's histidinyl ligands are not within this range, Gel filtration studies demonstrated that rcBphF readily oligomerizes in solution. Crystals of rcBphF were obtained using sodium formate or poly(ethylene glycol) (PEG) as the major precipitant. Analysis of the intermolecular contacts in the crystal revealed a head-to-tail interaction that occludes the cluster, but is very unlikely to be found in solution. Oligomerization of rcBphF in solution was reversed by the addition of dithiothreitol and is unrelated to the noncovalent crystallographic interactions. Moreover, the oligomerization state of rcBphF did not influence the latter's reduction potential. These results indicate that the 450 mV spread in reduction potential of Rieske clusters of dioxygenase-associated fi ferredoxins and mitochondrial bc(1) complexes is not due to significant differences in their solvent exposure.