Adir N, Axelrod H L, Beroza P, Isaacson R A, Rongey S H, Okamura M Y, Feher G
Department of Physics, University of California, San Diego, La Jolla, California 92093-0319 USA.
Biochemistry. 1996 Feb 27;35(8):2535-47. doi: 10.1021/bi9522054.
The photosynthetic reaction center (RC) of Rhodobacter sphaeroides and cytochrome c2 (cyt c2), its physiological secondary electron donor, have been co-crystallized. The molar ratio of RC/cyt c2 was found by SDS-PAGE and optical absorbance changes in the co-crystals to be 4. The crystals diffracted X-rays to 3.5 angstroms. However, the resolution degraded during data collection. A data set, 82.5% complete, was collected to 4.5 angstroms. The crystals belong to the tetragonal space group P4(3)2(1)2, with unit cell dimensions of a = b = 142.7 angstroms and c = 254.8 angstroms. The positions of the RCs in the unit cell were determined by molecular replacement. A comparable search for the cyt c2 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 c2 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 c2, 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 c2 amino acid residues. The RC-cyt c2 structure derived from the co-crystal data was supported by use of omit maps and structure refinement analyses. Cyt c2 reduces the photooxidized primary donor D+ in 0.9 +/- 0.1 micros 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 c2 complex: (i) Docking calculations based on interprotein electrostatic interactions identified possible binding positions of the cyt c2 on the RC. The cyt c2 position with the lowest electrostatic energy is very similar to that of the cyt c2 in the proposed co-crystal structure. (ii) Site-directed mutagenesis was used to modify two aspartic acid residues (M184 and L155) on the periplasmic surface of the RC. Cyt c2 binding affinity to these RCs and electron transfer rates to D+ in these RCs support the co-crystal structure of th RC-cyt c2 complex.
球形红杆菌的光合反应中心（RC）与其生理性二级电子供体细胞色素c2（cyt c2）已成功共结晶。通过SDS-PAGE以及共晶体中的吸光度变化，确定RC/cyt c2的摩尔比为4。这些晶体的X射线衍射分辨率可达3.5埃。然而，在数据收集过程中分辨率有所下降。最终收集到一个完整度为82.5%、分辨率为4.5埃的数据集。这些晶体属于四方晶系空间群P4(3)2(1)2，晶胞参数为a = b = 142.7埃，c = 254.8埃。通过分子置换确定了晶胞中RC的位置。由于细胞色素对总散射的贡献较小且占有率较低，用这种方法对cyt c2进行类似搜索未成功。通过手动将cyt c2定位到与RC的M多肽亚基周质表面相邻的差分电子密度区域。差分电子密度不足以精确确定cyt c2的位置，通过将血红素的暴露边缘朝向反应中心D的初级供体，并形成用于静电相互作用的RC和cyt c2氨基酸残基对来模拟其方向。通过使用省略图和结构精修分析，支持了从共晶体数据得出的RC-cyt c2结构。在共晶体中，cyt c2在0.9±0.1微秒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。这一结果有力地证明了共晶体中复合物的结构与溶液中的结构相同。还使用了另外两种方法来研究RC-cyt c2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 c2在RC上可能的结合位置。静电能最低的cyt c2位置与所提出的共晶体结构中的cyt c2位置非常相似。（ii）使用定点诱变来修饰RC周质表面的两个天冬氨酸残基（M184和L155）。cyt c2与这些RC的结合亲和力以及向这些RC中的D+的电子转移速率支持了RC-cyt c2复合物的共晶体结构。
