Gupta N, Bonomi F, Kurtz D M, Ravi N, Wang D L, Huynh B H
Department of Chemistry, University of Georgia, Athens 30602.
Biochemistry. 1995 Mar 14;34(10):3310-8. doi: 10.1021/bi00010a021.
The gene encoding Desulfovibrio (D.) vulgaris rubrerythrin (Prickril, B. C., Kurtz, D. M., Jr., LeGall, J., & Voordouw, G. (1991) Biochemistry 30, 1118), a protein of unknown function containing both FeS4 and (mu-oxo)diiron sites, was cloned and overexpressed in Escherichia coli. Upon cell lysis, the overexpressed protein was found in an insoluble form deficient in iron. Iron was incorporated in vitro by dissolving the protein in 3 M guanidinium chloride, adding Fe(II) anaerobically and diluting the denaturant. This recombinant rubrerythrin was found to have properties very similar to those of rubrerythrin isolated from D. vulgaris, except that the recombinant rubrerythrin contained six rather than four (or five) iron atoms per 44 kDa homodimer. Analyses of UV-vis, Mössbauer, and EPR spectra showed that the six iron atoms in recombinant rubrerythrin are organized as two FeS4 and two (mu-oxo/hydroxo)diiron sites. In order to allow examination of the diiron sites in the absence of the FeS4 sites, a truncated gene encoding the N-terminal 152 residues of D. vulgaris rubrerythrin was also cloned and overexpressed as an insoluble protein in E. coli, and iron was incorporated by a procedure analogous to that for recombinant rubrerythrin. This so-called "chopped" rubrerythrin (CRr) was found to consist of an approximately 35 kDa homodimer containing four iron atoms. Spectroscopic characterization indicated that the four iron atoms in CRr are organized as two diiron sites, the majority of which closely resemble the (mu-oxo)diiron(III) sites in E. coli ribonucleotide reductase R2 protein, and a minor fraction of which resemble the mixed-valent diiron(II,III) site in methane monooxygenase hydroxylase.(ABSTRACT TRUNCATED AT 250 WORDS)
编码脱硫弧菌（D.）普通红氧还蛋白（Prickril, B. C., Kurtz, D. M., Jr., LeGall, J., & Voordouw, G. (1991) Biochemistry 30, 1118）的基因被克隆并在大肠杆菌中过表达，该蛋白功能未知，含有FeS4和（μ-氧代）二铁位点。细胞裂解后，发现过表达的蛋白以不溶性形式存在且缺铁。通过将蛋白溶解在3 M氯化胍中，厌氧添加Fe(II)并稀释变性剂，在体外掺入铁。发现这种重组红氧还蛋白的性质与从普通脱硫弧菌中分离的红氧还蛋白非常相似，只是重组红氧还蛋白每44 kDa同型二聚体含有六个而非四个（或五个）铁原子。紫外可见光谱、穆斯堡尔光谱和电子顺磁共振光谱分析表明，重组红氧还蛋白中的六个铁原子组织成两个FeS4和两个（μ-氧代/羟基）二铁位点。为了在没有FeS4位点的情况下研究二铁位点，还克隆了编码普通脱硫弧菌红氧还蛋白N端152个残基的截短基因，并在大肠杆菌中作为不溶性蛋白过表达，通过类似于重组红氧还蛋白的方法掺入铁。发现这种所谓的“切碎”红氧还蛋白（CRr）由一个约3 kDa的同型二聚体组成，含有四个铁原子。光谱表征表明，CRr中的四个铁原子组织成两个二铁位点，其中大部分与大肠杆菌核糖核苷酸还原酶R2蛋白中的（μ-氧代）二铁（III）位点非常相似，一小部分与甲烷单加氧酶羟化酶中的混合价二铁（II,III）位点相似。（摘要截短至250字）
