Photocycle of halorhodopsin from Halobacterium salinarium.
Photocycle of halorhodopsin from Halobacterium salinarium.
复制标题
DOI：
10.1016/s0006-3495(95)80385-1
复制
发表时间：
1995-05
影响因子：
3.4
通讯作者：
G. Varo;L. Zimányi;Xiaolei Fan;Li Sun;R. Needleman;J. Lanyi
中科院分区：
文献类型：
--
作者：
G. Varo;L. Zimányi;Xiaolei Fan;Li Sun;R. Needleman;J. Lanyi
光驱动的氯离子泵，盐视紫红质，是一种混合物，含有全反式和13-顺式视网膜发色团在光和暗适应条件下，可以存在于无氯离子和氯离子结合的形式。为了描述与运输相关的全反式氯离子结合态的光化学循环，从而启动氯离子易位机制的研究，必须首先剖析这些物种对测量的光谱变化的贡献。我们解决了多个光化学反应，通过确定闪光灯诱导的差异光谱和光循环动力学在盐生盐杆菌制备的含盐视紫红质的膜，与光和暗适应的样品在不同的氯化物浓度。克隆的盐视紫红质的高表达使得使用未分级的细胞被膜进行这些测量成为可能，其中发色团不仅在氯化钠的存在下而且在用于参考的硫酸钠溶液中都是光稳定的。在选定的波长下仔细检查闪光引起的变化，可以将光谱变化分离成成分，并将其分配给各个光周期。根据研究结果，对H.盐藻盐视紫红质及其对氯化物的依赖性是必需的。全反式氯离子结合形式的循环由以下图式描述：HR-hv-->K<==>L1<==>L2<==>N-->HR，其中HR、K、L和N表示卤视紫红质及其光中间体。与早期的模型不同，这非常类似于当阻止席夫碱的去质子化时细菌视紫红质的光反应（例如，在低pH下或在D85 N突变体中）。也不像在早期的模型中，在这个光循环中没有一个步骤受到明显的影响时，氯离子浓度在20 mM和2 M之间变化，试图确定一个氯离子结合反应。
The light-driven chloride pump, halorhodopsin, is a mixture containing all-trans and 13-cis retinal chromophores under both light and dark-adapted conditions and can exist in chloride-free and chloride-binding forms. To describe the photochemical cycle of the all-trans, chloride-binding state that is associated with the transport, and thereby initiate study of the chloride translocation mechanism, one must first dissect the contributions of these species to the measured spectral changes. We resolved the multiple photochemical reactions by determining flash-induced difference spectra and photocycle kinetics in halorhodopsin-containing membranes prepared from Halobacterium salinarium, with light- and dark-adapted samples at various chloride concentrations. The high expression of cloned halorhodopsin made it possible to do these measurements with unfractionated cell envelope membranes in which the chromophore is photostable not only in the presence of NaCl but also in the Na2SO4 solution used for reference. Careful examination of the flash-induced changes at selected wavelengths allowed separating the spectral changes into components and assigning them to the individual photocycles. According to the results, a substantial revision of the photocycle model for H. salinarium halorhodopsin, and its dependence on chloride, is required. The cycle of the all-trans chloride-binding form is described by the scheme, HR-hv-->K<==>L1<==>L2<==>N-->HR, where HR, K, L, and N designate halorhodopsin and its photointermediates. Unlike the earlier models, this is very similar to the photoreaction of bacteriorhodopsin when deprotonation of the Schiff base is prevented (e.g., at low pH or in the D85N mutant). Also unlike in the earlier models, no step in this photocycle was noticeably affected when the chloride concentration was varied between 20 mM and 2 M in an attempt to identify a chloride-binding reaction.