VIBRATIONAL STUDIES OF ENERGY TRANSDUCING PROTEINS

这项研究的具体目的是描述反应中心(RC)蛋白中具有重要功能的辅因子的结构、振动和电子性质。主要的研究工具是共振拉曼光谱(RR)。第一个主要目标是表征具有特殊电子转移特性的转基因细菌RCS中的细菌叶绿素/细菌叶绿素(BCh1/BPH)的性质。遗传修饰包括在蛋白质的光物理活性L分支(即主要的电子受体)上替换初级电子供体(P)附近的副BChl和褐飞虱。这些研究主要集中在三类转基因RCS：(1)通过在BCh1/BPH大环V环附近添加/缺失氨基酸残基来改变BCh1L、BCh1M和BPhL附近的氢键相互作用和/或电场的突变体。这些研究的一个特别焦点涉及将(潜在的)带电残基放置在光活性辅因子附近的影响。(2)双(和更高阶)突变体，它将1类的替换特征整合到背景中，其中BPh1被BCh1分子(Beta型RCS)取代。(3)用非连接甘氨酸残基取代P的BChl的组氨酸轴向配体的突变体(空穴突变体)。在所有情况下，RR研究都将在RCS上进行，其详细的电子转移动力学已通过时间分辨光学实验阐明。第二个主要目标是进行RR研究，旨在完善光系统(PS)II RCS中放氧复合体的结构。这些研究的具体目标是直接参与分解水/析氧反应的锰团簇。重点将放在光谱的低频区域，在那里预计会发生锰-配体振动。为此，将获取其中已并入同位素标记(2H、18O、15N、37C1-/37C1-)和/或交换了诸如Ca+2和C1-等必需离子的PSII的RR数据(例如，用SR+2交换Ca+2或用Br-交换C1-)。细菌和PSII RCS研究的长期目标是确定辅因子的物理性质(结构、构象、电子密度分布)如何控制和/或反映它们的功能特征(跨越生物膜的电子转移/电荷分离；水分解/放氧)。

The specific aims of the proposed research are to characterize the structural, vibronic, and electronic properties of the functionally important cofactors in reaction center (RC) proteins. The principal investigative tool is resonance Raman (RR) spectroscopy. The first major objective is to characterize the properties of the bacteriochlorophyll/bacteriopheophytin (BCh1/BPh) in genetically modified bacterial RCs that exhibit unusual electron-transfer properties. The genetic modifications include replacements near the primary electron donor (P), both accessory BChls, and the BPh on the photophysically, active L branch of the protein (which is the primary electron acceptor). The studies focus on three general classes of genetically modified RCs: (1) Mutants in which the hydrogen bonding interactions and/or the electric fields in the vicinity of BCh1L, BCh1M, and BPhL are altered by addition/deletion of amino acid residues near ring V of the BCh1/BPh macrocycle. A particular focus of these studies concerns the effects of placing (potentially) charged residues near the photoactive cofactors. (2) Double (and higher order) mutants which incorporate the replacements characteristic of class 1 into a background in which BPhL is replaced with a BCh1 molecule (beta-type RCs). (3) Mutants in which the histidine axial ligands to the BChls of P are replaced by non-ligating glycine residues (cavity mutants). In all cases the RR studies will be conducted on RCs whose detailed electron-transfer kinetics have been elucidated via time-resolved optical experiments. The second major objective is to conduct RR studies aimed at refining the structure of oxygen- evolving complex in photosystem (PS) II RCs. The particular target of these studies is the manganese cluster, which directly mediates the water-splitting/oxygen-evolution reaction. The focus will be the low-frequency region of the spectrum where manganese-ligand vibrations are expected to occur. Toward this end, RR data will be acquired for PSII in which isotopic labels (2H, 18O, 15N, 37C1-/37C1- ) have been incorporated and/or essential ions such as Ca+2 and C1- have been exchanged (for example, Sr+2 for Ca+2 or Br- for C1-). The long-term objective of the studies on bacterial and PSII RCs is to determine how the physical properties of the cofactors (structure, conformation, electron-density distribution) govern and/or reflect their functional characteristics (electron transfer/charge separation across the biological membrane; water splitting/oxygen evolution).