Mechanism and Macromolecular Organization in Photosynthetic Reaction Centers

为了了解细菌光合反应中心初始电荷分离步骤的机制，提出了几个研究方向。(i)测量特殊对最低Qy电子态的共振拉曼强度和激发谱，结合光化学烧孔、吸收和电吸收（斯塔克效应）光谱学，获得激发态动力学信息；（ii）制备和表征特殊对附近的位点特异性突变体，这些突变体会扰乱其氧化还原特性，以获得可能影响单向电子转移的局部介电特性的信息；（iii）在外加电场中研究供体、受体和介导态氧化还原性质与野生型不同的RC突变体的量子产率和动力学，以确定快速电荷分离和慢电荷重组反应的机理。光合作用反应中心是最小的叶绿素-蛋白质复合物，能够维持光诱导的电荷分离反应，从而启动光合作用。第一反应是任何系统中已知的最快的反应之一。除了这些反应作为地球上储存化学能的主要来源的明显重要性之外，这些反应具有更广泛的生物物理和化学意义。这是因为电子转移反应在自然界中无处不在，而且由于这种反应相对简单，因此有可能在基本水平上理解反应机理。细菌反应中心是第一个用x射线晶体学表征的膜蛋白，因此我们所有的工作都属于结构-功能关系的总标题。本研究的重点是通过改变内部或外部电场来系统地改变反应中心的电荷转移反应速率和机理。内部场可以通过使用重组DNA方法改变特定的氨基酸或氨基酸的延伸来改变。外场既可以与电极一起应用于大块样品，也可以作为跨膜电位。后者在几乎所有的生物组装中都非常重要，我们试图了解将这些潜力与生物功能结合起来的机制。

In order to understand the mechanism of the initial charge separation steps in bacterial photosynthetic reaction centers, several lines of investigation are proposed. (i) Measurements of the resonance Raman intensities and excitation profile for the lowest Qy electronic state of the special pair, combined with photochemical holeburning, absorption and electroabsorption (Stark effect) spectroscopy, to obtain information on excited state dynamics; (ii) preparation and characterization of site-specific mutants in the vicinity of the special pair which perturb its redox properties to obtain information on the local dielectric properties which may affect unidirectional electron transfer; and (iii) studies of the quantum yields and kinetics of RC mutants whose donor, acceptor and mediating state redox properties are different from wild-type in applied electric fields to determine the mechanism of the fast charge separation and slower charge recombination reactions. %%% Photosynthetic reaction centers are the smallest chlorophyll- protein complex capable of sustaining the light-induced charge separation reactions which initiate photosynthesis. The first reactions are among the fastest reactions known in any system. In addition to the obvious importance of these reactions as the primary source of stored chemical energy on earth, these reactions have much broader biophysical and chemical significance. This is because electron transfer reactions are ubiquitous in nature, and because of the relative simplicity of this type of reaction, there is some prospect of being able to understand the reaction mechanism at a fundamental level. Bacterial reaction centers were the first membrane protein to be characterized by x-ray crystallography, and consequently all of our work falls under the general heading of structure-function relationships. The proposed research focuses on systematically altering the rates and mechanisms of charge-transfer reactions in the reaction center by modifying either internal or external electric fields. Internal fields can be altered by changing specific amino acids or stretches of amino acids using recombinant DNA methods. External fields can be applied either with electrodes to a bulk sample or as transmembrane potentials. The latter are of very great importance in nearly all biological assemblies and we seek to understand the mechanisms which couple such potentials to biological function.