Molecular cloning, overexpression in Escherichia coli, structural and functional characterization of house fly cytochrome b(5)
Molecular cloning, overexpression in Escherichia coli, structural and functional characterization of house fly cytochrome b(5)
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DOI：
10.1074/jbc.271.43.26637
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发表时间：
1996-10-25
影响因子：
4.8
通讯作者：
Feyereisen, R
中科院分区：
文献类型：
--
作者：
Guzov, VM;Houston, HL;Feyereisen, R
克隆了家蝇微粒体细胞色素b(5)基因，并对其进行了序列分析，全长家蝇细胞色素b(5)(134个氨基酸残基)的氨基酸序列与大鼠微粒体细胞色素b(5)的氨基酸序列同源性为48%。家蝇细胞色素b(5)蛋白在大肠杆菌中高效表达、纯化和鉴定。吸收光谱和EPR谱显示了与脊椎动物细胞色素b(5)非常相似的性质，核磁共振谱表明蛋白质中的血红素相对于其α，伽马介质轴的取向约为1：1。在六次铬(III)存在下，在修饰金电极上用循环伏安法测得标准氢电极的氧化还原电位为-26 mV。在重组体系中，家蝇细胞色素P450还原酶对细胞色素b(5)有较高的还原速率(5.5 S(-1))，当家蝇细胞色素P450还原酶、细胞色素P450 6A1、磷脂和洗涤剂组成的重组体系时，家蝇细胞色素b(5)能刺激七氯环氧化。在细胞色素P450 6A1浓度不变的情况下，细胞色素b(5)降低了P450还原酶的表观K-m，增加了七氯环氧化的V-max。结果表明，在细胞色素P4506A1周转过程中，细胞色素b(5)刺激了第一次电子转移之后的一步。
A microsomal cytochrome b(5) cDNA from the house fly, Musca domestica, was cloned and sequenced, The deduced amino acid sequence of the full-length house fly cytochrome b(5) (134 residues) is 48% identical to that of rat microsomal cytochrome b(5). The house fly cyto chrome b(5) protein was overexpressed in Escherichia coli, purified, and characterized. Absorption and EPR spectroscopy reveal properties very similar to cytochromes b(5) from vertebrates, NMR spectra indicate that the orientation of the heme in the protein relative to its alpha,gamma meso axis is about 1:1. A redox potential of -26 mV versus standard hydrogen electrode was measured by cyclic voltammetry on a modified gold electrode in the presence of hexamminechromium(III) chloride. The cytochrome b(5) is reduced by house fly cytochrome P450 reductase in a reconstituted system at a high rate (5.5 s(-1)), and it stimulates heptachlor epoxidation when res constituted with house fly cytochrome P450 reductase, cytochrome P450 6A1, phospholipid, and detergent. Cytochrome b(5) decreases the apparent K-m for P450 reductase and increases the V-max for heptachlor epoxidation at constant cytochrome P450 6A1 concentrations. The results indicate that cytochrome b(5) stimulates a step following the first electron transfer during cytochrome P450 6A1 turnover.