嗜热栖热菌细胞色素ba3亚基II的水溶性重组铜A结构域。
Water-soluble, recombinant CuA-domain of the cytochrome ba3 subunit II from Thermus thermophilus.
作者信息
Slutter C E, Sanders D, Wittung P, Malmström B G, Aasa R, Richards J H, Gray H B, Fee J A
机构信息
Division of Chemistry and Chemical Engineering, California Institute ofTechnology, Pasadena, 91125, USA.
出版信息
Biochemistry. 1996 Mar 19;35(11):3387-95. doi: 10.1021/bi9525839.
Recently, the genes of cytochrome ba3 from thermus thermophilus [Keightley, J.A., et al. (1995) J. Biol. Chem. 270, 20345-20358], a homolog of the heme-copper oxidase family, have been cloned. We report here expression of a truncated gene, encoding the copper A (CuA) domain of cytochrome ba3, that is regulated by a T7 RNA polymerase promoter in Escherichia coli. The CuA-containing domain is purified in high yields as a water-soluble, thermostable, purple-colored protein. Copper analysis by chemical assay, mass spectrometry, X-ray fluorescence, and EPR spin quantification show that this protein contains two copper ions bound in a mixed-valence state, indicating that the CuA site in cytochrome ba3, is a binuclear center. The absorption spectrum of the CuA site, free of the heme interference in cytochrome ba3, is similar to the spectra of other soluble fragments from the aa3-type oxidase of Parachccus denitrificans [Lappalainen, P., et al. (1993) J. Biol Chem. 268, 26416-26421] and the caa3-type oxidase of Bacillus subtilis [von Wachenfeldt, C. et al. (1994) FEBS Lett. 340, 109-113]. There are intense bands at 480 nm (3100 M(-1) cm(-1)) and 530 nm (3200 M(-1) cm(-1)), a band in the near -IR centered at 790 nm (1900 M(-1) cn(-1)), and a weaker band at 363 nm (1300M(-1) cm(-1)). The visible CD spectrum shows a positive-going band at 460 nm and a negative-going band at 527 nm, the opposite signs of which may result from the binuclear nature of the site. The secondary structure prediction from the far-UV CD spectrum indicates that this domain is predominantly beta-sheet, in agreement with the recent X-ray structure reported for the complete P. denitrificans cytochrome aa3 molecule [Iwata, S., et al. (1995) Nature 376, 660-669] and the engineered, purple CyoA protein [Wilmanns, M., et al. (1996) Proc. Natl Acad. Sci. U.S.A. 92, 11955-11959]. However, the thermostability of the fragment described here (Tm approximately 80 degrees C) and the stable binding of copper over a broad pH range (pH 3-9) suggest this protein may be uniquely suitable for detailed physical-chemical study.
最近，嗜热栖热菌中细胞色素ba3的基因[凯斯利，J.A.等人（1995年）《生物化学杂志》270卷，20345 - 20358页]，即血红素 - 铜氧化酶家族的一个同源物，已被克隆。我们在此报告一个截短基因的表达情况，该基因编码细胞色素ba3的铜A（CuA）结构域，它在大肠杆菌中受T7 RNA聚合酶启动子调控。含CuA的结构域以高产率被纯化，成为一种水溶性、热稳定的紫色蛋白质。通过化学分析、质谱、X射线荧光和电子顺磁共振（EPR）自旋定量进行的铜分析表明，这种蛋白质含有两个以混合价态结合的铜离子，这表明细胞色素ba3中的CuA位点是一个双核中心。CuA位点的吸收光谱，不受细胞色素ba3中血红素的干扰，与反硝化副球菌aa3型氧化酶[拉帕莱宁，P.等人（1993年）《生物化学杂志》268卷，26416 - 26421页]和枯草芽孢杆菌caa3型氧化酶[冯·瓦申费尔特，C.等人（1994年）《欧洲生物化学学会联合会快报》340卷，109 - 113页]的其他可溶性片段的光谱相似。在480纳米（3100 M⁻¹ cm⁻¹）和530纳米（3200 M⁻¹ cm⁻¹）处有强吸收带，在近红外区域以790纳米（1900 M⁻¹ cm⁻¹）为中心有一个吸收带，在363纳米（1300 M⁻¹ cm⁻¹）处有一个较弱的吸收带。可见圆二色光谱在460纳米处显示一个正向吸收带，在527纳米处显示一个负向吸收带，其相反的符号可能是由于该位点的双核性质所致。从远紫外圆二色光谱预测的二级结构表明，这个结构域主要是β折叠，这与最近报道的完整反硝化副球菌细胞色素aa3分子的X射线结构[岩田，S.等人（1995年）《自然》376卷，660 - 669页]以及工程化的紫色CyoA蛋白[威尔曼斯，M.等人（1996年）《美国国家科学院院刊》92卷，11955 - 11959页]一致。然而，这里描述的片段的热稳定性（熔点约80℃）以及在较宽pH范围（pH 3 - 9）内铜的稳定结合表明，这种蛋白质可能特别适合进行详细的物理化学研究。
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