Váró G, Zimányi L, Fan X, Sun L, Needleman R, Lanyi J K
Department of Physiology and Biophysics, University of California, Irvine 92717, USA.
Biophys J. 1995 May;68(5):2062-72. doi: 10.1016/S0006-3495(95)80385-1.
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.
光驱动氯离子泵——嗜盐菌视紫红质，在明适应和暗适应条件下均为包含全反式和13 - 顺式视黄醛发色团的混合物，并且能以无氯离子和结合氯离子的形式存在。为了描述与转运相关的全反式、结合氯离子状态的光化学循环，从而开启对氯离子转运机制的研究，首先必须剖析这些物质对所测光谱变化的贡献。我们通过测定来自盐生盐杆菌的含嗜盐菌视紫红质的膜在不同氯离子浓度下明适应和暗适应样品的闪光诱导差光谱和光循环动力学，解析了多个光化学反应。克隆的嗜盐菌视紫红质的高表达使得用未分级的细胞膜进行这些测量成为可能，在这种细胞膜中，发色团不仅在存在氯化钠时而且在用作参比的硫酸钠溶液中都是光稳定的。仔细检查选定波长处的闪光诱导变化，能够将光谱变化分离成组分并将它们归属于各个光循环。根据结果，需要对盐生盐杆菌嗜盐菌视紫红质的光循环模型及其对氯离子的依赖性进行实质性修订。全反式结合氯离子形式的循环由以下方案描述：HR - hv --> K <==> L1 <==> L2 <==> N --> HR，其中HR、K、L和N表示嗜盐菌视紫红质及其光中间体。与早期模型不同，这与在阻止席夫碱去质子化时（例如，在低pH或D85N突变体中）细菌视紫红质的光反应非常相似。同样与早期模型不同，当氯离子浓度在20 mM至2 M之间变化以试图确定一个氯离子结合反应时，这个光循环中的任何步骤都没有受到明显影响。
