Faculty of Pharmaceutical Sciences, Okayama University , Okayama 700-8530, Japan.
Atmosphere and Ocean Research Institute, The University of Tokyo , Chiba 277-8564, Japan.
J Am Chem Soc. 2017 Mar 29;139(12):4376-4389. doi: 10.1021/jacs.6b12139. Epub 2017 Mar 16.
In organisms, ion transporters play essential roles in the generation and dissipation of ion gradients across cell membranes. Microbial rhodopsins selectively transport cognate ions using solar energy, in which the substrate ions identified to date have been confined to monovalent ions such as H, Na, and Cl. Here we report a novel rhodopsin from the cyanobacterium Synechocystis sp. PCC 7509, which inwardly transports a polyatomic divalent sulfate ion, SO, with changes of its spectroscopic properties in both unphotolyzed and photolyzed states. Upon illumination, cells expressing the novel rhodopsin, named Synechocystis halorhodopsin (SyHR), showed alkalization of the medium only in the presence of Cl or SO. That alkalization signal was enhanced by addition of a protonophore, indicating an inward transport of Cl and SO with a subsequent secondary inward H movement across the membrane. The anion binding to SyHR was suggested by absorption spectral shifts from 542 to 536 nm for Cl and from 542 to 556 nm for SO, and the affinities of Cl and SO were estimated as 0.112 and 5.81 mM, respectively. We then performed time-resolved spectroscopic measurements ranging from femtosecond to millisecond time domains to elucidate the structure and structural changes of SyHR during the photoreaction. Based on the results, we propose a photocycle model for SyHR in the absence or presence of substrate ions with the timing of their uptake and release. Thus, we demonstrate SyHR as the first light-driven polyatomic divalent anion (SO) transporter and report its spectroscopic characteristics.
在生物体中,离子转运蛋白在跨细胞膜的离子梯度的产生和耗散中发挥着重要作用。微生物视紫红质利用太阳能选择性地转运同源离子,迄今为止,被鉴定的底物离子仅限于单价离子,如 H、Na 和 Cl。在这里,我们报告了一种来自蓝藻 Synechocystis sp. PCC 7509 的新型视紫红质,它可以向内转运多原子二价硫酸根离子 SO,其在未光解和光解状态下的光谱性质都会发生变化。在光照下,表达新型视紫红质的细胞,命名为 Synechocystis 盐藻紫质(SyHR),仅在存在 Cl 或 SO 的情况下使培养基碱化。该碱化信号通过添加质子载体得到增强,表明 Cl 和 SO 的内向转运伴随着随后膜内二次内向 H 运动。阴离子与 SyHR 的结合通过从 542nm 到 536nm(Cl)和从 542nm 到 556nm(SO)的吸收光谱位移来暗示,Cl 和 SO 的亲和力分别估计为 0.112mM 和 5.81mM。然后,我们进行了从飞秒到毫秒时间域的时间分辨光谱测量,以阐明光反应过程中 SyHR 的结构和结构变化。基于这些结果,我们提出了一个在不存在或存在底物离子的情况下,SyHR 光反应的循环模型,以及它们的摄取和释放时间。因此,我们证明了 SyHR 是第一个光驱动的多原子二价阴离子(SO)转运体,并报告了其光谱特征。