【作者】 陈晓波；

【导师】 匡廷云； 李良璧；

【作者基本信息】 中国科学院研究生院（植物研究所） ， 植物学， 2006， 博士

【摘要】 细胞色素b6f蛋白复合体(Cytochrome b6f complex, Cyt b6f)是光合膜上参与光合作用原初反应过程的主要膜蛋白超分子复合体之一。莱茵衣藻和嗜热蓝细菌的Cyt b6f三维晶体结构均显示,每Cyt b6f单体分子含有1分子Chlorophyll a (Chl a ),充分肯定了Chl a是Cyt b6f天然成分的观点(Kurisu et al,2003;Stroebel et al,2003)。研究表明不同来源的Cyt b6f中Chla单线激发态寿命(或荧光寿命)并不一样,多数的研究结果认为Cyt b6f中Chla单线激发态寿命只有200ps左右,但是也有Cyt b6f中Chla单线激发态寿命为～600ps的报道;而甲醇中游离Chl a的单线激发态寿命为4ns左右。针对Cyt b6f中Chla单线激发态寿命快速淬灭的现象,Dashdorj等(2005)根据晶体结构推测Cyt b6f中Chla单线激发态和邻近的Cyt b6亚基上Tyr105残基发生电子交换传递,从而快速淬灭Chla单线激发态,减少了三线态Chl a和单线态氧的产生,并且认为这是Cyt b6f保护自身不受单线态氧破坏的一种机制,但是这一推测缺乏有力的证据。另外,Cyt b6f中Chla的功能仍然未知。本文以菠菜Cyt b6f为对象,结合多种实验手段,测定了菠菜Cyt b6f中Chl a单线激发态寿命,并对复合体中Chl a单线激发态淬灭的机理进行了深入研究。此外,我们还对复合体中Chl a可能的功能进行了初步地探讨。获得了如下的结果:1.针对不同来源的Cyt b6f中Chla单线激发态寿命(或荧光寿命)测定结果不同的报道,仔细分析了其中的原因,发现除了样品来源的差异外,使用不同的去垢剂可能是一个不可忽视的因素。在实验中,不同的研究者分别采用了十二烷基麦芽糖苷(n-Dodecylβ-D-maltoside,DDM,)和八烷基葡萄糖苷(n-Octylβ-D-glucopyranoside,β-OG)作为溶解样品的去垢剂。因此,本文借助稳态吸收和稳态荧光光谱、瞬态光散射技术,CD光谱和亚皮秒时间分辨吸收光谱等技术,分别研究了这两种去垢剂对Cyt b6f结构和功能的不同影响。结果表明,DDM去垢剂能使Cyt b6f处于较好的分散体系中,其中血红素和Chl a分子处于特定的蛋白环境中,不会导致Cyt b6f变性;而β-OG去垢剂会使Cyt b6f产生聚合现象,其中的血红素和Chl a与蛋白环境的相互作用减弱,和DDM相比其电子传递活性显著降低,Chl a单线激发态寿命延长,Chl a更容易被光更多还原

【Abstract】 The cytochrome b6f (Cyt b6f) complex is one of the three integrate membrane protein complex in the photosynthetic electron transport chain. According to the X-ray structure analysis (Kurisu et al, 2003; Stroebel et al, 2003), one chlorophyll a (Chl a ) molecule is bound between helices F and G of subunit IV of this complex. The Chl a in the complex, however, has a smaller excited-state life time, most probably dued to quenching by the electron exchange between the Chl a and nearby amino acid(Tyr105 of Cyt b6), than that of free Chl a in methanol(~4ns), and thus reduces the formation of the Chl a triplet state (Dashdorj et al, 2005). This is considered as one kind of protection mechanism of the Chl a against singlet oxygen in the complex although there are not any evidences for the mechanism still. Also, the role(s) of this Chl a remains unclear. In this work, the excited-state life time of Chl a in the Cyt b6f from spinach was determined, the quenching mechanism of the excited state of Chl a in the complex was investigated, and the function of the Chl a was also proposed based on some experiments data.1. Different excited-state life times of Chl a in different Cyt b6f sources were obtained in some reports. But the two detergents, n-Dodecylβ-D-maltoside (DDM) and n-Octylβ-D-glucopyranoside (β-OG),were used respectively in the reports. So the effects of the two detergents on the structure and function of the Cyt b6f were investigated by stead-state absorption, fluorescence, CD, dynamic light scattering, and time-resolved absorption spectroscopy in this work firstly. The results are as follows: the DDM detergent dissolves the Cyt b6f better thanβ-OG detergent does; DDM can keep the Cyt b6f in native state, under which the hemes and Chl a experience specific environment, while theβ-OG make the complex in denatured state in some sort, under which the catalyzed electron transfer activity of the complex更多还原