Primary Electron Transfer Processes in Photosynthetic Bacterial Reaction Centers

细菌RC是一种高度对称的蛋白质，具有两条色素链，称为L和M分支，本质上是彼此的镜像。然而，电荷分离仅通过L分支(分别为BL、HL和QA)上的细菌叶绿素、细菌叶绿素和苯醌辅助因子进行。多年来，M支链颜料(BM、HM和QB-)的作用，或者更具体地说，如何实现电子转移到L支链的“单向性”，一直是该领域的一个焦点。最近，本实验室通过靶向改变BL、HL和BM附近的氨基酸，实现了~23%的电子转移到RC中正常不活跃的M分支。目前的工作试图在这一进展的基础上，以及在理解沿光活性分支电荷分离高产率的因素方面取得的平行和同等重要的进展的基础上。具体地说，将在RC蛋白中进行进一步的定点突变，并对其对所有主要事件的影响进行全面研究，以实现以下目标。(1)阐明每个辅助因子参与初级事件，它们参与的机制，以及相关电荷分离态的自由能关系如何影响电子转移的方向性以及电荷分离相对于电荷复合的速率和产额。(2)了解极性和可电离氨基酸残基如何调节附近辅因子的电子和振动性质，从而调节光化学。(3)推导出两个支链上的相对自由能和电子耦合对方向性的贡献(例如，涉及P*和P+BL-与P+BM-)。(4)电子转移到M支链(形成P+HM-)和沿该支链转移(形成P+QB-)的产额比迄今所获得的更大。这将为P+QB的研究开辟许多令人兴奋的新途径--通过M侧与L侧形成的P+QB，以及双方的事件是如何通过类似的突变来操纵的。地球上的生命最终依赖于光合作用，光合作用是植物和某些细菌将太阳光能量转化为化学势能的过程。这种转化通过一系列快速的电子转移反应发生，这些反应在称为反应中心(RC)的特殊膜结合的色素-蛋白质复合体中分离电荷。值得注意的是，在RC中，电荷分离是通过两种可能的途径之一单向进行的，量子产率为~100%。本研究以紫色光合细菌的RCS为研究对象，试图在分子水平上对这一初级电荷分离过程有一个全面的认识。为此，将使用跨越飞秒(10-15秒)到几秒时间尺度的瞬时吸收光谱测量以及其他光谱技术来直接探测电荷分离的不同阶段以及中间体的形成和衰变速度。这些研究将为最重要的生物过程之一的分子水平机制提供进一步的详细见解，有助于开发用于太阳能转换的仿生学，并对生物物理学中更广泛的主题产生积极影响，包括生物电子转移和蛋白质在调节其辅因子功能特性中的作用。

HoltenMCB 0077187The bacterial RC is a highly symmetric protein that has two chains of pigments, referred to as the L and M branches that are essentially mirror images of one another. However, charge separation takes place exclusively via the bacteriochlorophyll, bacteriopheophytin, and quinone cofactors on the L branch (BL, HL, and QA, respectively). The role of the M branch pigments (BM, HM, and QB-), or more specifically how "unidirectionality" of electron transfer to the L branch is achieved, has been a focus of the field for many years. Recently, this laboratory has achieved an ~23% yield of electron transfer to the normally inactive M branch in a RC through targeted site-directed changes in the amino acids near BL, HL, and BM. The current work seeks to build on this progress as well as on parallel and equally important advances in understanding the factors underpinning the high yield of charge separation along the photoactive branch. Specifically, further site-directed mutations will be made in the RC protein and comprehensive studies of their effects on all the primary events carried out in order to achieve the following. (1) Elucidation of the participation of each cofactor in the primary events, the mechanisms of their involvement, and how the free-energy relationships of the associated charge-separated states impact the directionality of electron transfer and the rates and yields of charge separation versus charge recombination. (2) Understanding the manner in which polar and ionizable amino acid residues modulate the electronic and vibrational properties of the nearby cofactors and, thus, the photochemistry. (3) Deducing the contributions to directionality of the relative free energies and electronic couplings on the two branches (e.g. involving P* and P+BL- versus P+BM-). (4) Obtaining a greater yield of electron transfer both to the M branch (forming P+HM-) and along this branch (forming P+QB-) than has been achieved to date. This will open up many new and exciting avenues for work on P+QB- formed via the M-side versus the L-side and how the events on the two sides are manipulated by analogous mutations. Life on earth ultimately is dependent on photosynthesis, the process by which plants and certain bacteria convert the energy of sunlight into chemical potential energy. This conversion occurs via a series of fast electron transfer reactions that separate charge in a specialized membrane-bound pigment-protein complex called the reaction center (RC). Remarkably, charge separation in the RC takes place unidirectionally via one of two possible pathways and with a quantum yield of ~100%. This research project focuses on RCs from purple photosynthetic bacteria and seeks to come to a comprehensive molecular level understanding of this primary charge separation process. To this end, transient absorption spectroscopic measurements spanning the femtosecond (10-15 sec) to seconds time scales as well as other spectroscopic techniques will be used to directly probe the various stages of charge separation and the rates of formation and decay of the intermediates. These studies will provide further detailed insights into the molecular-level mechanism of one of the most important biological processes, aid in the development of biomimetics for solar energy conversion, and positively impact broader topics in biophysics including biological electron transfer and the role of a protein in modulating the functional properties of its cofactors.