Mutational Analysis of Photosystem I Function

细胞中的壳聚糖能量的产生需要跨膜的电子传递，这通常是通过多蛋白酶来调节的，例如呼吸作用的细胞色素氧化酶和光合作用的光系统。光系统I是蓝藻和叶绿体光合作用电子传递链的两个膜结合反应中心之一。它的功能类似于光驱动的质蓝蛋白-铁氧还蛋白氧化还原酶，是一种异源多聚体色素-蛋白质复合体。在这个项目中，PI试图通过定点突变和靶向基因失活来确定光系统I蛋白的功能。光系统I复合体包含两个叶醌分子，其中一个(或两个)作为氧化还原中心A1。PI建议通过诱变在叶醌结合部位附近引入半胱氨基残基，并给它们贴上自旋标记。标记和A1信号之间的顺磁相互作用将使我们能够识别具有氧化还原活性的叶醌(S)，从而识别电子转移途径的活性分支。PI已经产生了在其光系统I复合体中含有叶Q的突变体。PI建议使用这些突变体在体内A1位招募外源苯二酚。在与A1位的结合中成功地与天然质醌竞争的苯二酚将有助于确定在极低的氧化还原电位下对其功能重要的苯醌的结构特征。不能替代叶醌功能的苯二酚将被用于结合部位的定向进化，以容纳和使用这些苯醌。除了研究光系统I中的叶素外，PI还将研究b-胡萝卜素分子在光系统I中激发能量转移中的作用。PI最近发现一些光系统I蛋白被翻译后修饰。他建议识别这些修饰并研究它们的功能意义。电子转移反应是光合作用、呼吸作用、药物代谢和许多其他生化途径的关键步骤。光合作用和呼吸作用包含具有普遍意义的膜结合电子转移链。能量传递复合体中的电子转移蛋白提供结构稳定性，识别反应伙伴，并准确定位辅因子。我们期望拟议的研究结果将提供有关蛋白质环境如何决定辅因子功能特性的信息。此外，研究结果还将提出修改蛋白质以适应和利用外来辅因子的方法，以及设计更好的仿生光转化系统的方法。植物、藻类和蓝藻中的产氧光合作用是地球上生命的主要能源和氧气来源。因此，有必要更好地了解这一过程，以应对未来的环境和粮食挑战。

0078264ChitnisEnergy generation in a cell requires electron transfer across membranes, which is typically mediated through multiprotein enzymes, such as cytochrome oxidase of respiration and photosystems of photosynthesis. Photosystem I is one of the two membrane-bound reaction centers of the photosynthetic electron transfer chain in cyanobacteria and chloroplasts. It functions as the light-driven plastocyanin-ferredoxin oxidoreductase and is a heteromultimeric pigment-protein complex. In this project, the PI is attempting to determine the function of photosystem I proteins through site-directed mutagenesis and targeted gene inactivation. A photosystem I complex contains two phylloquinone molecules, one (or both) of which serves as the redox center A1. The PI proposes to introduce cysteinyl residues near the phylloquinone-binding site by mutagenesis and to attach spin labels to them. Paramagnetic interactions between the label and A1 signal will allow identification of the redox-active phylloquinone(s) and consequently the active branch of electron transfer pathway. The PI has generated phylloquinone-less mutants that contain plastoquinone in their photosystem I complexes. The PI proposes to use those mutants to recruit foreign quinones in the A1 site in vivo. The quinones that compete successfully with the native plastoquinone in binding to the A1 site will be useful in determining structural features of the quinones that are important for their function at an extremely low redox potential. The quinones that are unable to substitute function of phylloquinone will be used for directed evolution of the binding site to accommodate and to use these quinones. In addition to studies on phylloquinones in photosystem I, the PI will investigate the role of b-carotene molecules in the excitation energy transfer in photosystem I. The PI recently discovered that several photosystem I proteins are modified post-translationally. He proposes to identify these modifications and examine their functional significance.Electron transfer reactions are key steps in photosynthesis, respiration, drug metabolism, and many other biochemical pathways. Photosynthesis and respiration contain membrane-bound electron transfer chains of general significance. The electron-transfer proteins in energy-transducing complexes provide structural stability, recognize reaction partners, and accurately orient cofactors. We expect that the outcome of the proposed research will provide information about how the protein environment determines functional properties of cofactors. In addition, the results will suggest ways to modify proteins for accommodating and using foreign cofactors and to engineer better biomimetic photoconversion systems. Oxygenic photosynthesis in plants, algae and cyanobacteria is the primary source of energy and oxygen for the life on our planet. Therefore, better understanding of this process is necessary to address the environmental and food challenges of the future.