Vidakovic M S, Fraczkiewicz G, Germanas J P
Department of Chemistry, University of Houston, Houston, Texas 77204-5641, USA.
J Biol Chem. 1996 Jun 21;271(25):14734-9. doi: 10.1074/jbc.271.25.14734.
The heterologous expression and spectroscopic characterization of the [2Fe-2S] ferredoxin from the sexually transmitted human parasite Trichomonas vaginalis is described. Using oligonucleotide primers based on the deduced DNA sequence, the gene encoding the ferredoxin was amplified by polymerase chain reaction and cloned into a T7 RNA polymerase expression vector. Expression of the gene in Escherichia coli host HMS174(DE3) resulted in the high level production of the protein with the correctly assembled iron-sulfur cluster. The absorption, circular dichroism, resonance Raman, and EPR spectra of the recombinant protein revealed many differences from those of other [2Fe-2S] ferredoxins. The redox potential of the protein (-347 mV versus normal hydrogen electrode) was also determined. Whereas the amino acid sequence of T. vaginalis ferredoxin showed greatest homology to the [2Fe-2S] ferredoxins found in bacteria and vertebrate mitochondria which function in cytochrome P450 oxidation pathways, the spectroscopic properties showed substantial dissimilarity. Differences in the biophysical properties and function of T. vaginalis ferredoxin are proposed to result from the characteristic amino acid sequence of the parasite protein near the cysteine residues that ligate the valence-localized Fe(III) site of the reduced cluster.
本文描述了来自性传播人类寄生虫阴道毛滴虫的[2Fe-2S]铁氧化还原蛋白的异源表达及光谱表征。利用基于推导的DNA序列设计的寡核苷酸引物，通过聚合酶链反应扩增编码铁氧化还原蛋白的基因，并将其克隆到T7 RNA聚合酶表达载体中。该基因在大肠杆菌宿主HMS174(DE3)中的表达导致了具有正确组装的铁硫簇的蛋白质的高水平产生。重组蛋白的吸收光谱、圆二色光谱、共振拉曼光谱和电子顺磁共振光谱显示出与其他[2Fe-2S]铁氧化还原蛋白的许多差异。还测定了该蛋白的氧化还原电位（相对于标准氢电极，为-347 mV）。虽然阴道毛滴虫铁氧化还原蛋白的氨基酸序列与在细胞色素P450氧化途径中起作用的细菌和脊椎动物线粒体中的[2Fe-2S]铁氧化还原蛋白具有最大的同源性，但其光谱性质却有很大不同。有人提出，阴道毛滴虫铁氧化还原蛋白的生物物理性质和功能的差异是由寄生虫蛋白在连接还原簇的价态定位Fe(III)位点的半胱氨酸残基附近的特征氨基酸序列所致。
