Sommer Frederik, Drepper Friedel, Hippler Michael
Lehrstuhl für Biochemie der Pflanzen, Institut für Biologie II, Universität Freiburg, Schänzlestrasse 1, 79104 Freiburg, Germany.
J Biol Chem. 2002 Feb 22;277(8):6573-81. doi: 10.1074/jbc.M110633200. Epub 2001 Dec 14.
At the lumenal side of photosystem I (PSI) in cyanobacteria, algae, and vascular plants, proper recognition and binding of the donor proteins plastocyanin (pc) and cytochrome (cyt) c(6) are crucial to allow subsequent efficient electron transfer to the photooxidized primary donor. To characterize the surface regions of PSI needed for the correct binding of both donors, loop j of PsaB of Chlamydomonas reinhardtii was modified using site-directed mutagenesis and chloroplast transformation. Mutant strains D624K, E613K/D624K, E613K/W627F, and D624K/W627F accumulated <20% of PSI as compared with wild type and were only able to grow photoautotrophically at low light intensities. Mutant strains E613N, E613K, and W627F accumulated >50% of PSI as compared with wild type. This was sufficient to isolate the altered PSI and perform a detailed analysis of the electron transfer between the modified PSI and the two algal donors using flash-induced spectroscopy. Such an analysis indicated that residue Glu(613) of PsaB has two functions: (i) it is crucial for an improved unbinding of the two donors from PSI, and (ii) it orientates the positively charged N-terminal domain of PsaF in a way that allows efficient binding of pc or cyt c(6) to PSI. Mutation of Trp(627) to Phe completely abolishes the formation of an intermolecular electron transfer complex between pc and PSI and also drastically diminishes the rate of electron transfer between the donor and PSI. This mutation also hinders binding and electron transfer between the altered PSI and cyt c(6). It causes a 10-fold increase of the half-time of electron transfer within the intermolecular complex of cyt c(6) and PSI. These data strongly suggest that Trp(627) is a key residue of the recognition site formed by the core of PSI for binding and electron transfer between the two soluble electron donors and the photosystem.
在蓝细菌、藻类和维管植物中，光系统I（PSI）的腔面，供体蛋白质体蓝素（pc）和细胞色素（cyt）c6的正确识别与结合对于随后有效地将电子传递给光氧化的初级供体至关重要。为了表征PSI中两个供体正确结合所需的表面区域，利用定点诱变和叶绿体转化对莱茵衣藻PsaB的环j进行了修饰。与野生型相比，突变株D624K、E613K/D624K、E613K/W627F和D624K/W627F积累的PSI不到20%，并且仅能在低光照强度下进行光合自养生长。与野生型相比，突变株E613N、E613K和W627F积累的PSI超过50%。这足以分离出改变的PSI，并使用闪光诱导光谱对修饰的PSI与两种藻类供体之间的电子传递进行详细分析。这样的分析表明，PsaB的Glu(613)残基有两个功能：（i）它对于改善两个供体从PSI上的解离至关重要，（ii）它以一种允许pc或cyt c6有效地与PSI结合的方式使PsaF带正电荷的N末端结构域定向。将Trp(627)突变为Phe完全消除了pc与PSI之间分子间电子传递复合物的形成，并且也极大地降低了供体与PSI之间的电子传递速率。这种突变也阻碍了改变的PSI与cyt c6之间的结合和电子传递。它导致cyt c6与PSI分子间复合物内电子传递半衰期增加10倍。这些数据有力地表明，Trp(627)是由PSI核心形成的识别位点的关键残基，用于两种可溶性电子供体与光系统之间的结合和电子传递。
