Ta D T, Vickery L E
Department of Physiology and Biophysics, University of California, Irvine 92717.
J Biol Chem. 1992 Jun 5;267(16):11120-5.
Escherichia coli contains a soluble, [2Fe-2S] ferredoxin of unknown function (Knoell, H.-E., and Knappe, J. (1974) Eur. J. Biochem. 50, 245-252). Using antiserum to the purified protein to screen E. coli genomic expression libraries, we have cloned a gene (designated fdx) encoding this protein. The DNA sequence of the gene predicts a polypeptide of 110 residues after removal of the initiator methionine (polypeptide M(r) = 12,186, holoprotein M(r) = 12,358). The deduced amino acid sequence is strikingly similar to those of the ferredoxins found in animal mitochondria which function with cytochrome P450 enzymes and to the ferredoxin from Pseudomonas putida which functions with P450cam. The overall sequence identity is approximately 36% when compared with human mitochondrial and P. putida ferredoxins, and the identities include 4 cysteine residues proposed to coordinate the iron cluster. The protein was overproduced approximately 500-fold using an expression plasmid, and the holoprotein was assembled and accumulated in amounts exceeding 30% of the total cell protein. The overexpressed ferredoxin exhibits absorption, circular dichroism, and electron paramagnetic resonance spectra closely resembling those of the animal ferredoxins and P. putida ferredoxin.
大肠杆菌含有一种功能未知的可溶性[2Fe-2S]铁氧化还原蛋白（克内尔，H.-E.，和克纳普，J.（1974年）《欧洲生物化学杂志》50，245 - 252）。利用针对纯化蛋白的抗血清筛选大肠杆菌基因组表达文库，我们克隆了一个编码该蛋白的基因（命名为fdx）。该基因的DNA序列预测去除起始甲硫氨酸后有一个110个残基的多肽（多肽M(r)=12,186，全蛋白M(r)=12,358）。推导的氨基酸序列与在动物线粒体中与细胞色素P450酶一起发挥作用的铁氧化还原蛋白以及恶臭假单胞菌中与P450cam一起发挥作用的铁氧化还原蛋白的序列惊人地相似。与人类线粒体和恶臭假单胞菌的铁氧化还原蛋白相比，总体序列同一性约为36%，并且同一性包括被认为用于配位铁簇的4个半胱氨酸残基。使用表达质粒使该蛋白过量表达约500倍，并且全蛋白组装并积累，其含量超过总细胞蛋白的30%。过量表达的铁氧化还原蛋白表现出与动物铁氧化还原蛋白和恶臭假单胞菌铁氧化还原蛋白非常相似的吸收、圆二色性和电子顺磁共振光谱。
