Molecular insights into evolution and mechanisms of binding, release and electron transfer between plastocyanin or cytochrome c6 and photosystem I

绿藻中的质蓝蛋白(PC)或细胞色素C6(CytC6)与光系统I(PSI)之间的电子转移反应为了解蛋白质-蛋白质识别的一般原理提供了一个独特的系统。这是一个很有吸引力的分子模型，用来研究在体外和生理条件下如何优化伙伴之间的电子转移，其中反应伙伴的浓度、pH值、溶液的离子强度等参数是永久变化的基础。我们将对PSI亚基psaA/PsaB和核编码的cytc6以及胞体和细胞核的转化和异源表达进行定点突变，以改变对供体和PSI之间的最佳结合/释放和电子转移至关重要的特定残基。电子转移反应将在体外和体内通过快速吸收光谱以及化学交联技术和质谱学进行分析。我们的目标是从机制上深入了解(I)结合、释放和电子转移是如何在进化过程中优化的，(Ii)提供一个详细的分子模型，(Iii)了解这些优化是如何反映在藻类的生理性能中的。

The electron transfer reaction between plastocyanin (pc) or cytchrome c6 (cyt c6) and photosystem I (PSI) in green algae provides a unique system to gain molecular insights in general principles of protein-protein recognition. It is an attractive molecular model to investigate how electron transfer is optimized between partners in vitro and under physiological in vivo conditions where concentration of reaction partners, the pH value, the ionic strength of the solution and other parameter underlie permanent changes. We will perform site-directed mutagenesis of the plastid-encoded genes for PSI subunits PsaA/PsaB and nuclear encoded cyt c6 as well as plastid and nuclear transformation and heterologous expression of cyt c6 to alter specific residues that are important for optimal binding/ release and electron transfer between the donors and PSI. The electron transfer reaction will be analysed in vitro and in vivo by fast absorbance spectroscopy as well as chemical crosslinking techniques and mass spectrometry. It is our goal to provide indepth mechanistic understanding of how (i) binding, release and electron transfer were optimized during evolution, (ii) to provide a detailed molecular model and (iii) understand how these optimizations are reflected in the physiological performance of the alga.