Primary Electron Transfer Processes in Photosynthetic Bacterial Reaction Centers

HoltenMCB 0077187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，它们基本上是彼此的镜像。 然而，电荷分离仅通过L分支上的细菌叶绿素、细菌脱镁叶绿素和醌辅因子（分别为BL、HL和QA）发生。 M分支颜料（BM、HM和QB-）的作用，或更具体地说，如何实现电子转移到L分支的“单向性”，多年来一直是该领域的焦点。 最近，该实验室通过对BL、HL和BM附近的氨基酸进行靶向定点改变，实现了~23%的电子转移至RC中通常无活性的M分支。 目前的工作旨在建立在这一进展以及平行和同样重要的进展，在理解的因素支撑沿着光活性分支的电荷分离的高产率。具体而言，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)阐明每个辅因子参与的主要事件，其参与的机制，以及相关的电荷分离状态的自由能关系如何影响电子转移的方向性和电荷分离与电荷重组的速率和产量。 (2)了解极性和可电离的氨基酸残基调节附近辅因子的电子和振动特性的方式，从而调节光化学。 (3)推导两个分支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)获得了比迄今为止所获得的更大的电子转移到M分支（形成P+HM-）和沿着该分支（形成P+QB-）的产率。 这将为P+QB的工作开辟许多新的和令人兴奋的途径-通过M侧与L侧形成，以及两侧的事件如何被类似的突变操纵。地球上的生命最终依赖于光合作用，这是植物和某些细菌将阳光能量转化为化学势能的过程。 这种转换是通过一系列快速的电子转移反应发生的，这些反应将电荷分离到一个称为反应中心（RC）的专门的膜结合色素蛋白复合物中。 值得注意的是，RC中的电荷分离通过两种可能的途径之一单向发生，量子产率为~ 100%。 本研究项目的重点是从紫色光合细菌的RC，并寻求来一个全面的分子水平上了解这一主要的电荷分离过程。 为此，跨越飞秒（10-15秒）至秒时间尺度的瞬态吸收光谱测量以及其他光谱技术将用于直接探测电荷分离的各个阶段以及中间体的形成和衰减速率。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详细的见解，帮助开发用于太阳能转换的仿生学，并积极影响生物物理学中更广泛的主题，包括生物电子转移和蛋白质在调节其辅助因子的功能特性中的作用。

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.