CAREER: Manipulating Directionality of Electron Transfer Within Type 1 Photosynthetic Reaction Centers

光合作用反应中心（RCs）是生命中最古老、最有用的装置之一，它使生物圈能够利用不断撞击地球的丰富太阳能，并使物种多样化，形成大量具有不同生物能量策略的物种。所有已知的RCs都具有对称结构，使用两个相似或相同的整体膜亚基形成二聚体核心，结合辅因子，电子通过辅因子在膜上转移。这种对称的排列产生了两个类似的辅助因子分支，光驱动的电子转移可以沿着这两个分支进行。每个分支的前两个成员是氯，而第三个成员是醌。众所周知，在具有良好特征的2型RCs中，初始电子转移几乎完全沿着两个分支中的一个发生，尽管这种强不对称性的起源仍然存在争议。光系统I （PS1）仍然是1型RCs的最佳表征代表，但PS1中电子转移方向的许多方面仍然未知。最近的光学研究清楚地表明，在环境温度下，电子转移可以利用PS1的两个辅助因子分支，而电子顺磁共振（EPR）数据表明，在低温下，只有一个分支是活跃的。本项目旨在探索PS1中电子双向转移的性质、起源和程度。这将主要通过对PS1核心多肽的遗传操作来实现，以使电子在两个途径之间的分配偏倚。操作的效果将通过时间分辨光学和EPR光谱进行评估，这应该能够区分PS1每个分支中的叶绿醌分子，从而提供每个途径相对使用的评估。主电子供体和受体附近的突变可能以可预测的方式影响ET在两条通路上发生的程度。外部条件和温度在决定这两个分支的利用中的作用也将被探讨。最终，长期目标是推导出自然界用于指导光驱动电子转移的一般规则。这项工作的更广泛的影响包括培养研究生和本科生在化学、生物学和物理学的交叉科学领域。它将加强几个研究小组之间的联系，特别是雷丁小组和巴黎生物物理化学研究所（IBPC, Paris）的Fabrice Rappaport和Pierre Joliot小组。此外，这一特殊的国际合作将通过一名博士后的参与而扩大，他将花相当多的时间在IBPC，使用他们的仪器。该项目将有助于在阿拉巴马大学建立EPR设施，该设施已经吸引了附近几个研究所的合作努力。最后，获得的知识和专业知识可能具有实际意义，例如重新设计rc的能力，以便两个分支之间的划分对外部刺激（例如小离子，疏水配体的结合，电场等）做出反应，并且可以以受控的方式进行指导。

Photosynthetic reaction centers (RCs) are one of life's most ancient and useful devices, allowing the biosphere to exploit the abundant solar energy continuously striking our planet and to diversify into a huge number of species with distinct bioenergetic strategies. All known RCs have symmetric structures, using two similar or identical integral membrane subunits to form a dimeric core, which binds the cofactors through which electrons are transferred across the membrane. This symmetric arrangement gives rise to two similar branches of cofactors down which light-driven electron transfer could proceed. The first two members of each branch are chlorins, while the third is a quinone. It is known that the initial electron transfer occurs almost exclusively along one of the two branches in the well-characterized type 2 RCs, although the origins of this strong asymmetry are still debated. Photosystem I (PS1) is still the best-characterized representative of the type 1 RCs, but many aspects of the direction of electron transfer in PS1 remain unknown. Recent optical work clearly suggests that electron transfer can make use of both cofactor branches of PS1 at ambient temperature, while electron paramagnetic resonance (EPR) data indicate that only one branch is active at low temperature. The purpose of this project is to explore the nature, origins, and degree of bi-directionality of electron transfer in PS1. This will be accomplished mainly by genetic manipulation of the PS1 core polypeptides to bias the partitioning of electrons between the two pathways. The effect of the manipulations will be assessed by time-resolved optical and EPR spectroscopy, which should be capable of distinguishing between the phylloquinone molecules in each branch of PS1, thus providing an assessment of the relative use of each pathway. Mutations near the primary electron donor and acceptors may influence the degree to which ET occurs down the two pathways in predictable ways. The role of external conditions and temperature in determining the utilization of the two branches will also be explored. Ultimately, the long-term goal is to deduce the general rules used by nature to direct light-driven electron transfer.Broader impacts of this work include the training of graduate students and undergraduate students in interdisciplinary science at the interface of chemistry, biology, and physics. It will strengthen ties between several research groups, especially the Redding group and that of Fabrice Rappaport and Pierre Joliot at the Institut de Biologie Physico-Chimique (IBPC, Paris). Moreover, this particular international collaboration will be enlarged by the engagement of a postdoctoral fellow who will spend a considerable amount of time at the IBPC, using their instruments. This project will help to build up the EPR facilities at the University of Alabama, which has already attracted collaborative efforts from several nearby institutes. Finally, the knowledge and expertise gained may have practical aspects, such as the ability to re-engineer RCs, so that partitioning between the two branches becomes responsive to external stimuli (e.g. small ions, binding of hydrophobic ligands, electric field, etc.) and may be directed in a controlled way.