Investigation of the functional mechanisms of artificial photosynthetic pigment-protein supra-molecular complexes

三种不同的色素蛋白复合物（LH2和LH1两个触角和反应中心RC）深入参与了紫色光合细菌的初级过程。为了更深入地了解这些色素-蛋白复合物的功能机制，本课题对利用有机合成修饰类胡萝卜素色素结构的人工色素-蛋白复合物进行了广泛的研究。实现了以下显著特点：1。实现了类似类胡萝卜素的大规模全合成：实现了一系列具有不同缀合度的球状胡萝卜素、螺旋黄质和番茄红素的类似类胡萝卜素的大规模全合成。类胡萝卜素类似物被掺入反应中心的x射线晶体学：球形红杆菌的类胡萝卜素类似物被掺入无类胡萝卜素反应中心（RC）。用x射线晶体学方法对制备的RCs进行了结晶和晶体结构测定。这些结构分析揭示了当类胡萝卜素分子被纳入RC时，其单向插入。PheM162是一个看门氨基酸残基，而TrpM75是一个锁氨基酸残基，当类胡萝卜素分子重新掺入R26.1 rc中时。天然LH2天线配合物的电吸收光谱：采用双相锁定检测的电吸收光谱被认为是检测色素-蛋白质配合物中色素与载脂蛋白之间静电相互作用的最有前途的方法之一。建立了准确的实验方案和分析方法，并将其应用于光合细菌Rb， sphaeroides菌株gic的天然LH2复合物。重新加入类似类胡萝卜素的人工反应中心的电吸收光谱：电吸收光谱应用于Rb的无类胡萝卜素反应中心（RC）。sphaeroides菌株R26.1以及含有类似类胡萝卜素的相同RC。我们第一次定量地确定了与rc结合的细菌叶绿素分子周围存在或不存在类胡萝卜素分子所引起的静电环境的差异。类胡萝卜素的时间分辨吸收光谱：将时间分辨光谱技术应用于f3-胡萝卜素和番茄红素。本文首次在实时域对单光子允许的1^1B_u^+(S_2)和禁止的2^1Ag^-(S_1)态之间的中间单重态激发态（Sx态）进行了时间解析，并证明了该中间激发态参与了光激发到1^1B_u^+态的弛豫过程。本研究扩展到具有不同多烯链长度的胡萝卜素分子系列。除了具有5个共轭C=C键的胡萝卜素分子外，所有胡萝卜素分子在从S_2到S_1.6的弛豫过程中都具有S_x态。红毛Rs. rubrum LHI天线复合物的重组及其电吸收光谱分析：用纯化的全反式螺旋黄质重组红毛Rs. rubrum菌株G9+ B880色素蛋白复合物的无类胡萝卜素单体亚基，形成人工光收获1（LH1）复合物。利用电吸收光谱对重组后的LH1进行分析，以评价复合物中色素与载脂蛋白之间的静电相互作用。少

Three different kinds of pigment-protein complexes (two antennae called LH2 and LH1, and reaction center (RC)) are deeply involved in the primary process of purple photosynthetic bacteria. In this research project, for the deeper understanding of the functional mechanisms of these pigment-protein complexes, artificial pigment-protein complexes in which the structure of the carotenoid pigments were modified by utilizing organic synthesis were extensively studied. The following remarkable features have been achieved.1.Achievement of the large-scale total synthesis of analogue carotenoids : Larges-scale total syntheses of a series of analogue carotenoids of spheroidene, spirilloxanthin, and lycopene which have different extent of conjugation were achieved.2.X-ray crystallography of the reaction centers in which the analogue carotenoids have been incorporated : The analogue carotenoids of spheroidene were incorporated into the carotenoidless reaction center (RC) from a photosynthetic bacte … More rium Rhodobacter sphaeroides strain R26.1. Thus prepared artificial RCs were crystallized and their crystal structures were determined by X-ray crystallography. These structure analyses revealed the unidirectional insertion of the carotenoid molecule when it is incorporated into the RC. PheM162 is a gate-keeper amino-acid residue, while TrpM75 is a locking amino-acid residue when the carotenoid molecule is re-incorporated into the R26.1 RC.3.Electroabsorption spectroscopy on the native LH2 antenna complex : Electroabsorption spectroscopy using dual-phase lock-in detection was proposed to be one of the most promising methods to detect the electrostatic interaction between the pigments and apoproteins in the pigment-protein complexes. Exact experimental protocols together with the analysis methods were constructed, and they were applied to the native LH2 complex from a photosynthetic bacterium Rb, sphaeroides strain GIC.4.Electroabsorption spectroscopy of the artificial reaction centre in which an analogue carotenoid has been re-incorporated : Electroabsorption spectroscopy was applied to the carotenoidless reaction centre (RC) from Rb. sphaeroides strain R26.1 as well as the same RC in which an analogue carotenoid has been incorporated. We have, for the first time, quantitatively determined the difference of the electrostatic environment induced by the presence or absence of a carotenoid molecule around the bacteriochlorophyll molecules bound to the RC.5.Sub-20 fs time-resolved absorption spectroscopy of carotenoids : Time-resolved absorption spectroscopy with unprecedented temporal resolution of sub-20 fs was applied to f3-carotene and lycopene. The intermediate singlet excited-state (Sx state) in-between the one-photon allowed 1^1B_u^+(S_2) and forbidden 2^1Ag^-(S_1) states was time-resolved for the first time in the real time-domain, and it was evidenced that this intermediate excited state was involved in the relaxation process following the photoexcitation up to the 1^1B_u^+ state. This study was extended to the series of carotene molecules which have different polyene chain-lengths. Except the case of the carotene molecule having 5 conjugated C=C bonds, all the carotene molecules were evidenced in real time-domain to have the S_x state in the relaxation process from S_2 to S_1.6.Reorganization of the LHI antenna complex of Rs. rubrum and its electroabsorption spectroscopy : Carotenoid-free monomeric subunits of B880 pigment-protein complex from Rs. rubrum strain G9+ was reorganized with purified all-trans-spirilloxanthin to form the artificial light-harvesting 1(LH1) complex. Electroabsorption spectroscopy was applied to thus reconstituted LH1 in order to evaluate electrostatic interaction between pigments and apo-proteins in the complex. Less