Co-crystallization and characterization of the photosynthetic reaction center-cytochrome c(2) complex from Rhodobacter sphaeroides
Co-crystallization and characterization of the photosynthetic reaction center-cytochrome c(2) complex from Rhodobacter sphaeroides
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DOI：
10.1021/bi9522054
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发表时间：
1996-02-27
期刊：
影响因子：
2.9
通讯作者：
Feher, G
中科院分区：
文献类型：
--
作者：
Adir, N;Axelrod, HL;Feher, G
将球形红细菌的光合反应中心（RC）与其生理次级电子供体细胞色素c（2）（cyt c（2））共结晶。经SDS-PAGE分析，共晶体中RC/cyt c（2）的摩尔比为4。晶体衍射X射线至3.5埃。然而，在数据收集期间，分辨率降低。将82.5%完整的数据集收集到4.5埃。晶体属于四元晶系空间群P4（3）2（1）2，晶胞尺寸为a = B = 142.7埃，c = 254.8埃。RC在晶胞中的位置通过分子置换来确定。用这种方法对细胞色素c（2）进行类似的搜索是不成功的，因为细胞色素对总散射的贡献很小，而且它的占有率很低。将cyt c（2）手动定位到不同电子密度的斑块中，邻近RC的M多肽亚基的周质表面。不同的电子密度不足以精确定位cyt c（2），通过将血红素暴露的边缘朝向反应中心D的主要供体，并通过形成静电相互作用的RC和cyt c（2）氨基酸残基对，来模拟其取向。通过使用省略图和结构精修分析，支持从共晶数据导出的RC-cyt c（2）结构。Cyt c（2）在共晶体中在0.9 +/- 0.1 μ s内还原光氧化的初级供体D+，这与体内和溶液中的快速电子转移速率相同。这一结果提供了强有力的证据，证明复合物在共晶中的结构与在溶液中的结构相同。另外两种方法用于研究RC-cyt c（2）复合物的结构：（i）基于蛋白质间静电相互作用的对接计算确定了cyt c（2）在RC上的可能结合位置。具有最低静电能量的cyt c（2）位置与所提出的共晶结构中的cyt c（2）位置非常相似。(ii)使用定点诱变来修饰RC周质表面上的两个天冬氨酸残基（M184和L155）。Cyt c（2）与这些RC的结合亲和力和这些RC中D+的电子转移速率支持RC-cyt c（2）复合物的共晶结构。
The photosynthetic reaction center (RC) of Rhodobacter sphaeroides and cytochrome c(2) (cyt c(2)), its physiological secondary electron donor, have been co-crystallized. The molar ratio of RC/cyt c(2) was found by SDS-PAGE and optical absorbance changes in the co-crystals to be 4. The crystals diffracted X-rays to 3.5 Angstrom. However, the resolution degraded during data collection. A data set, 82.5% complete, was collected to 4.5 Angstrom. The crystals belong to the tetragonal space group P4(3)2(1)2, with unit cell dimensions of a = b = 142.7 Angstrom and c = 254.8 Angstrom. The positions of the RCs in the unit cell were determined by molecular replacement. A comparable search for the cyt c(2) by this method was unsuccessful because of the small contribution of the cytochrome to the total scattering and because of its low occupancy. The cyt c(2) was positioned manually into patches of difference electron density, adjacent to the periplasmic surface of the M polypeptide subunit of the RC. The difference electron density was not sufficient for precise positioning of the cyt c(2), and its orientation was modeled by placing the exposed edge of the heme toward the primary donor of the reaction center D and by forming pairs for electrostatically interacting RC and cyt c(2) amino acid residues. The RC-cyt c(2) structure derived from the co-crystal data was supported by use of omit maps and structure refinement analyses. Cyt c(2) reduces the photooxidized primary donor D+ in 0.9 +/- 0.1 mu s in the co-crystals, which is the same as the fast electron transfer rate in vivo and in solution. This result provides strong evidence that the structure of the complex in the co-crystal is the same as in solution. Two additional methods were used to investigate the structure of the RC-cyt c(2) complex: (i) Docking calculations based on interprotein electrostatic interactions identified possible binding positions of the cyt c(2) on the RC. The cyt c(2) position with the lowest electrostatic energy is very similar to that of the cyt c(2) in the proposed co-crystal structure. (ii) Site-directed mutagenesis was used to modify two aspartic acid residues (M 184 and L155) on the periplasmic surface of the RC. Cyt c(2) binding affinity to these RCs and electron transfer rates to D+ in these RCs support the co-crystal structure of the RC-cyt c(2) complex.