Remelli R, Varotto C, Sandonà D, Croce R, Bassi R
Università di Verona, Biotecnologie Vegetali, Strada Le Grazie, 37134, Verona, Italy.
J Biol Chem. 1999 Nov 19;274(47):33510-21. doi: 10.1074/jbc.274.47.33510.
The chromophore binding properties of the higher plant light-harvesting complex II have been studied by site-directed mutagenesis of pigment-binding residues. Mutant apoproteins were overexpressed in Escherichia coli and then refolded in vitro with purified chromophores to yield holoproteins selectively affected in chlorophyll-binding sites. Biochemical and spectroscopic characterization showed a specific loss of pigments and absorption spectral forms for each mutant, thus allowing identification of the chromophores bound to most of the binding sites. On these bases a map for the occupancy of individual sites by chlorophyll a and chlorophyll b is proposed. In some cases a single mutation led to the loss of more than one chromophore indicating that four chlorophylls and one xanthophyll could be bound by pigment-pigment interactions. Differential absorption spectroscopy allowed identification of the Q(y) transition energy level for each chlorophyll within the complex. It is shown that not only site selectivity is largely conserved between light-harvesting complex II and CP29 but also the distribution of absorption forms among different protein domains, suggesting conservation of energy transfer pathways within the protein and outward to neighbor subunits of the photosystem.
通过对色素结合残基进行定点诱变，研究了高等植物光捕获复合物II的发色团结合特性。突变载脂蛋白在大肠杆菌中过量表达，然后在体外与纯化的发色团重折叠，以产生在叶绿素结合位点受到选择性影响的全蛋白。生化和光谱表征显示每个突变体的色素和吸收光谱形式有特定损失，从而能够鉴定与大多数结合位点结合的发色团。基于这些结果，提出了叶绿素a和叶绿素b在各个位点占据情况的图谱。在某些情况下，单个突变导致不止一种发色团损失，这表明四个叶绿素和一个叶黄素可能通过色素 - 色素相互作用结合。差分吸收光谱法能够鉴定复合物中每个叶绿素的Q(y)跃迁能级。结果表明，不仅光捕获复合物II和CP29之间的位点选择性在很大程度上是保守的，而且不同蛋白质结构域之间吸收形式的分布也是保守的，这表明蛋白质内部以及向光系统相邻亚基的能量转移途径是保守的。
