紫细菌B800缺失LH2能量传递模型的构建及性质

Abstract

构建b800缺失lH2对于阐明光合作用中光能传递的分子机制与捕光复合体组装机制具有重要意义。采用吸收光谱、荧光光谱、分子筛层析、超滤和SdS-PAgE等方法研究了紫细菌两个典型种外周捕光复合体(lH2)约800nM特征光谱(b800)细菌叶绿素(bCHl)缺失能量传递模型的构建方法及性质。结果表明:在PH 8.0TrIS-HCl(10MMOl·l-1)缓冲液中,0.08%SdS能够使来自rHOdObACTEr AzOTOfOrMAnS的lH2b800bCHl特异性解离,解离体系中加入10%(φ)甲醇,通过超滤脱除游离bCHl,构建了b800缺失lH2,但该缺失模型不够稳定。在PH 1.9缓冲液中,来自rHOdOPSEudOMOnAS PAluSTrIS的lH2b800bCHl能够特异性解离,通过层析得到两个组分。一个组分的b800bCHl不能通过层析脱除,能够重新自组装成lH2。另一个组分为b800缺失lH2,该缺失模型稳定。两种lH2均存在2类以上b800bCHl结合位点,并得到了两类rHOdOPSEudOMOnAS PAluSTrIS b800bCHl解离的lH2,但未发现类似紫色硫细菌中的b800吸收光谱劈裂现象。b800缺失lH2均未呈现约800nM特征荧光光谱。采用两种方法构建了两个物种b800缺失lH2能量传递模型。利用bCHl与缺失b800lH2结合能力不同的特性,将rHOdOPSEudOMOnAS PAluSTrIS中的lH2分成两个类型,实现了异质性亚基lH2的分离。

To seek microscopic molecular mechanism of energy transfer and complex reconstitution in the photosynthesis,the conditions for construction of B850-only peripheral light-harvesting complex(LH2)and their properties were investigated using absorption,fluorescence spectroscopy,molecular sieve chromatography,ultrafiltration and sodium dodecylsulfate polyacrylamide gel electrophoresis(SDS-PAGE)from the purple bacteria.The results indicated that bacteriochlorophylls(BChl)of B800 incubated in 10mmo·L-1 Tris-HCl(pH 8.0)buffer are selectively released from their binding sites of LH2 of Rhodobacter azotoformans(A-LH2)by 0.08%(W/V)SDS.B850-only A-LH2 was constructed after removing free BChl mixing with 10% methyl alcohol by ultrafiltration.B850 BChl was released after A-LH2 was incubated for 240 min in dark at room temperature(RT).While BChl of B800 incubated in pH 1.9buffer were selectively released from their binding sites of LH2 of Rhodopseudomonas palustris(P-LH2).The authors acquired two components using molecular sieve chromatography.Free BChl of one component was not removed and self-assembled to P-LH2.The other removed free BChl and B850-only P-LH2 was constructed.B850 unchanged after P-LH2 was incubated.P-LH2αandβsubunits have different molecular weights,but those of A-LH2 are in the contrary.It is concluded that B850-only P-LH2 is more stable than A-LH2.The enigmatic split of the B800 absorption band was not observed in these LH2,but we acquired two kinds of B800-released LH2 from Rhodopseudomonas palustris.The authors' results may provide a new light to separate homogeneous Apoprotein LH2.