Geerts D, Schubert H, de Vrieze G, Borrias M, Matthijs H C, Weisbeek P J
Department of Molecular Cell Biology, University of Utrecht, The Netherlands.
J Biol Chem. 1994 Nov 11;269(45):28068-75.
The petE gene encoding plastocyanin precursor protein from the cyanobacterium Anabaena PCC 7937 was introduced in the cyanobacterial host strain Synechococcus PCC 7942. The host normally only uses cytochrome c553 as Photosystem I (PS I) donor. The heterologous gene was efficiently expressed using the inducible Escherichia coli trc promoter. Accumulation of plastocyanin protein depended on the presence of Cu2+. The protein was accurately targeted to the thylakoid lumen, from which it could be isolated in the mature form. Redox difference spectroscopy proved the presence of a Cu2+ ion in the holoenzyme. Isolated heterologous plastocyanin was functional in reconstitution of in vitro electron transfer to PS I. The presence of Anabaena plastocyanin in Synechococcus thylakoid membranes increased PS I electron transfer rate 2.5 times. Analysis of P700 redox and PS II fluorescence transients in vivo showed a faster electron transfer through PS I because of enhanced electron supply in the presence of plastocyanin. In addition, the distribution of electrons between photosynthetic and respiratory electron transfer changed. Plastocyanin preferentially donates electrons to PS I rather than to the respiratory cytochrome-c oxidase complex and is not functionally equivalent to cytochrome c553.
将来自蓝藻鱼腥藻PCC 7937的编码质体蓝素前体蛋白的petE基因导入蓝藻宿主菌株聚球藻PCC 7942中。该宿主通常仅使用细胞色素c553作为光系统I（PS I）供体。使用可诱导的大肠杆菌trc启动子高效表达异源基因。质体蓝素蛋白的积累取决于Cu2+的存在。该蛋白被准确地靶向到类囊体腔中，可从其中以成熟形式分离出来。氧化还原差光谱法证明全酶中存在Cu2+离子。分离出的异源质体蓝素在体外电子传递至PS I的重建中具有功能。聚球藻的类囊体膜中存在鱼腥藻质体蓝素使PS I电子传递速率提高了2.5倍。体内P700氧化还原和PS II荧光瞬态分析表明，由于质体蓝素存在时电子供应增强，通过PS I的电子传递更快。此外，光合和呼吸电子传递之间的电子分布发生了变化。质体蓝素优先将电子捐赠给PS I而不是呼吸细胞色素c氧化酶复合物，并且在功能上不等同于细胞色素c553。
