Active ion transport across membranes is vital to maintaining the electrochemical gradients of ions in cells and is mediated by transmembrane proteins. Halorhodopsin (HR) functions as a light-driven inward pump for chloride ions. The protein contains all-trans-retinal bound to a specific lysine residue through a protonated Schiff base. Interaction between the bound chloride ion and the protonated Schiff base is crucial for ion transport because chloride ion movement is driven by the flipping of the protonated Schiff base upon photoisomerization. However, it remains unknown how this interaction evolves in the HR photocycle. Here, we addressed the effect of the bound anion on the structure and dynamics of HR from Natronomonas pharaonis in the early stage of the photocycle. Comparison of the chloride-bound, formate-bound, and anion-depleted forms provided insights into the interaction between the bound anion and the chromophore/protein moiety. In the unphotolyzed state, the bound anion affects the π-conjugation of the polyene chain and the hydrogen bond of the protonated Schiff base of the retinal chromophore. Picosecond time scale measurements showed that the band intensities of the W16 and W18 modes of the tryptophan residues decreased instantaneously upon photoexcitation of the formate-bound form. In contrast, these intensity decreases were delayed for the chloride-bound and anion-depleted forms. These observations suggest the stronger interactions of the bound formate ion with the retinal chromophore and the chromophore pocket. On the nanosecond to microsecond timescales, we found that the interaction between the protonated Schiff base and the bound ion is broken upon formation of the K intermediate and is recovered following translocation of the bound anion toward the protonated Schiff base in the L intermediate. Our results demonstrate that the hydrogen-bonding ability of the bound anion plays an essential role in the ion transport of light-driven anion pumps.

中文翻译：

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结合阴离子对法老氏梭菌（Natronomonas pharaonis）卤代视紫红质的结构和动力学的影响。

跨膜的主动离子转运对于维持细胞内离子的电化学梯度至关重要，并由跨膜蛋白介导。卤素视紫红质（HR）用作光驱动的氯离子内向泵。该蛋白质包含通过质子化席夫碱与特定赖氨酸残基结合的全反式视网膜。结合的氯离子与质子化席夫碱之间的相互作用对于离子传输至关重要，因为氯离子的移动是由光异构化时质子化席夫碱的翻转驱动的。但是，仍然不清楚这种相互作用在HR光周期中如何演变。在这里，我们讨论了在光循环的早期，结合阴离子对法老对虾Natronomonas pharaonis HR的结构和动力学的影响。氯化物结合，甲酸结合，阴离子耗尽形式为结合阴离子与生色团/蛋白质部分之间的相互作用提供了见识。在未光解状态下，结合的阴离子影响视网膜发色团的多烯链的π共轭和质子化席夫碱的氢键。皮秒级的时标测量显示，色氨酸残基的W16和W18模式的能带强度在甲酸酯结合形式的光激发后立即降低。相反，对于结合氯离子和贫阴离子的形式，这些强度下降被延迟了。这些观察结果表明结合的甲酸盐离子与视网膜发色团和发色团袋之间的相互作用更强。在纳秒到微秒的时间尺度上，我们发现，质子化席夫碱与键合离子之间的相互作用在形成K中间体时被破坏，并在L型中间体中将键合阴离子向质子化席夫碱转移后被恢复。我们的结果表明，键合阴离子的氢键结合能力在光驱动阴离子泵的离子迁移中起着至关重要的作用。