Kataoka K, Nakai M, Yamaguchi K, Suzuki S
Department of Chemistry, Graduate School of Science, Osaka University, Japan.
Biochem Biophys Res Commun. 1998 Sep 18;250(2):409-13. doi: 10.1006/bbrc.1998.9310.
The gene coding for the 109-amino-acid, non-glycosylated form of mavicyanin was synthesized and expressed in Escherichia coli. The recombinant protein refolded from E. coli inclusion bodies was purified and characterized. Its spectroscopic properties are fully identical to those of mavicyanin isolated from zucchini, even in the absence of its carbohydrate moiety. The blue cooper center of mavicyanin strongly binds three ligands (2His and Cys) as well as many blue copper proteins. To disclose the fourth ligand of mavicyanin, Met was substituted for Gln95 by site-directed mutagenesis. The replacement changes from a rhombic EPR signal to an axial one and exhibits the quite similar absorption and CD spectra to those of plastocyanin. The midpoint potential of Gln95-->Met mavicyanin shows the positive shift of 187 mV compared with the recombinant protein, being close to the values of plastocyanins. The differences of the spectroscopic and electrochemical properties between mavicyanin and its mutant demonstrate that the fourth ligand of mavicyanin is Gln95.
编码109个氨基酸的非糖基化形式的马维花青素的基因被合成并在大肠杆菌中表达。从大肠杆菌包涵体中重折叠的重组蛋白被纯化并进行了表征。即使在没有碳水化合物部分的情况下，其光谱性质与从西葫芦中分离出的马维花青素完全相同。马维花青素的蓝色铜中心强烈结合三个配体（2个组氨酸和半胱氨酸）以及许多蓝色铜蛋白。为了揭示马维花青素的第四个配体，通过定点诱变将谷氨酰胺95替换为甲硫氨酸。这种替换使菱形电子顺磁共振信号变为轴向信号，并呈现出与质体蓝素非常相似的吸收光谱和圆二色光谱。与重组蛋白相比，谷氨酰胺95→甲硫氨酸马维花青素的中点电位显示正移187毫伏，接近质体蓝素的值。马维花青素与其突变体之间光谱和电化学性质的差异表明，马维花青素的第四个配体是谷氨酰胺95。
