Primary Electron Transfer Processes in Photosynthetic Bacterial Reaction Centers

这项研究的最终目标是实现对细菌光合作用反应中心(RC)光化学过程的统一分子水平的理解。细菌和植物的RCS都以潜在电子载体的两个准对称分支为特征。在细菌RC中，只有L分支(也称为A分支)被用于跨膜光致电子传递。在本地RC中，M分支(也称为B分支)处于非活动状态。该项目最近的工作描述了可以诱导M-分支辅助因子支持完全跨膜电子转移的条件，尽管产率很低。这一进展为详细探索天然RC中单向电子转移的起源以及沿着RC辅因子整个通常不活跃的M-分支的电子转移打开了大门。这些研究是本研究的基础。这项工作将通过操纵和研究通常不活跃的M分支上的相同过程，将现有的沿着本征RC中L侧电荷分离的高量子产额模型进行最终的一致性检验。这些发现将有助于阐明RC中作为一个统一整体的电子转移的机制基础。从更广泛的角度来看，拟议的工作将使通常不活跃的M侧辅助因子链以一种稳健的方式完全可用于许多实验室的各种下一代研究。这包括探索新的中间体和与QA/QB双电子门函数相关的构象或质子态的新途径，这些态通常不会在L侧电荷分离的正常过程中产生或仅瞬时形成。一个目标是操纵L和M侧电荷分离态的辅因子含量和自由能，以给通常不活跃的M-支链提供非常高的(接近定量的)电子转移产额。第二个目标将是探索电子耦合和能量学对方向性的贡献(即初始L侧与B侧电荷分离)。第三个目标是提高从M侧细菌叶绿素中间电子受体到末端醌受体QB的电子转移的速度和产量，并在这样做的过程中测试目前对类似的L侧过程的理解。这些目标将通过对RC突变体的静态和时间分辨(飞秒至秒)光谱研究来实现，以推导出电荷分离和复合过程的速率和产率以及参与态的相对自由能。这项研究是理解细菌RC中主要光化学事件的核心，并将继续为正在进行的绿色植物光系统I和II的研究提供指南。更广泛的影响：这项研究将继续在本科生和代表性不足的群体参与研究和科学以及研究人员接受广泛的多学科培训方面产生明显的影响。通过三个项目，将研究理念融入教学和教育发展将继续是合作伙伴关系的重点。(1)将为本科生或高中生量身定做网络教程，主题为“为什么草是绿的，血是红的？”(2)将广泛升级两个本科生物理化学实验，以探索叶绿素和相关发色团的光谱，以及光合作用的关键能量/电子传递过程，从面向应用的角度教授分子电子光谱和动力学。(3)密苏里州植物园成人教育推广计划的一部分，将开发一个广泛的、以科学为基础的讲座，内容涉及光合作用的一般和基本方面，并与园艺兴趣相联系。尽管光合作用的相关性是不可否认的，但该计划还没有这样的讲座/教育部分。最后，除了传统的传播手段(出版物、会议)外，还将通过与科学作家的接触，继续利用地方和国家流行的印刷品和网络出版场所，向广大个人传播光合作用研究(以及在该项目下作出的贡献)。

The ultimate goal of this research is to achieve a unified molecular level understanding of the photochemical processes in the bacterial photosynthetic reaction center (RC). The RCs of both bacteria and plants feature two quasi-symmetric branches of potential electron carriers. In the bacterial RC, only the L-branch (also called the A branch) is utilized for trans-membrane photo-induced electron transport. In the native RC, the M-branch (also called the B branch) is inactive. Recent work from this project has delineated conditions wherein the M-branch cofactors can be induced to support full trans-membrane electron transfer, albeit in low yield. This advance has opened the door for detailed explorations of the origins of the unidirectional electron transfer in the native RC, and of electron transfer along the entire normally inactive M-branch of RC cofactors. Such studies are the basis of this research. This work will put current models for the high quantum yield of charge separation along the L-side in the native RC to the ultimate test of consistency via manipulation and study of the same processes along the normally inactive M branch. The findings will help to elucidate the mechanistic underpinnings of electron transfer in the RC as a unified whole. From a wider perspective, the proposed work will make the normally inactive M-side cofactor chain fully accessible in a robust manner for a variety of next-generation studies in many laboratories. This includes new avenues for exploring novel intermediates and conformational or protonation states associated with the QA/QB two-electron gate function that are normally not produced or formed only transiently in the normal course of L-side charge separation. One goal will be to manipulate the cofactor content and the free energies of both the L and M-side charge-separated states to give a very high (approaching quantitative) yield of electron transfer to the normally inactive M-branch. A second goal will be to probe the contribution of electronic couplings along with energetics to directionality (i.e., initial L-side versus B-side charge separation). A third goal will be to increase the rate and yield of electron transfer from the M-side bacteriopheophytin intermediary electron acceptor to the terminal quinone acceptor QB, and in doing so test current understanding of the analogous L side processes. These goals will be pursued via static and time-resolved (femtoseconds to seconds) spectroscopic studies of RC mutants to derive the rates and yields of the charge separation and recombination processes and the relative free energies of the participating states. This research is central to understanding the primary photochemical events in the bacterial RC, and will continue to provide guideposts for research being conducted on photosystems I and II from green plants. Broader Impacts:This research will continue to have a demonstrated impact in the participation of undergraduates and underrepresented groups in research and science, and in the broad multidisciplinary training the researchers receive. The integration of research ideas into teaching and educational development will continue to be a focus of the Co-PIs, via three projects. (1) A web-based tutorial will be developed that can be tailored for undergraduate or high school students on the general topic "Why is grass green and blood red?" (2) Two undergraduate physical-chemistry laboratory experiments will be extensively upgraded to exploit the spectroscopy of chlorophyll and related chromophores, and the energy/electron transfer processes critical to photosynthesis, to teach molecular electronic spectroscopy and kinetics from application-oriented perspectives. (3) A broad, science-based lecture on general and fundamental aspects of photosynthesis with links to interests in gardening will be developed to be part of an adult education outreach program at the Missouri Botanical Garden. Despite the undeniable relevance of photosynthesis, the program has not had such a lecture/education component. Finally, in addition to the traditional means of dissemination (publications, meetings), local and national popular print and web-based publication venues will continue to be exploited through contacts with science writers in order to disseminate photosynthesis research (and contributions made under this project) to a broad spectrum of individuals.