Krokinobacter rhodopsin 2 (KR2) was discovered as the first light-driven sodium pumping rhodopsin (NaR) in 2013, which contains unique amino acid residues on C-helix (N112, D116, and Q123), referred to as an NDQ motif. Based on the recent X-ray crystal structures of KR2, the sodium transport pathway has been investigated by various methods. However, due to complicated structural information around the protonated Schiff base (PRSB) region in the dark state and lack of structural information in the intermediates with sodium bound in KR2, detailed sodium pump mechanism is still unclear. Here we applied comprehensive low-temperature light-induced difference FTIR spectroscopy on isotopically labeled KR2 WT and site-directed mutant proteins (N112A, D116E, R109A, and R109K). We assigned the N-D stretching vibration of the PRSB at 2095 cm-1 and elucidate the hydrogen bonding interaction with D116 (a counter ion for the PRSB). We also assigned strongly hydrogen-bonded water (2333 cm-1) near R109 and D251, and found that presence of a positive charge at the position of R109 is prerequisite for the pumping function of KR2.

中文翻译：

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红外光谱分析在光驱动钠泵KR2中基于视网膜异构化的质子化席夫碱周围的结构变化。

在2013年发现了Krokinobacter视紫红质2（KR2）作为第一个光驱动钠泵浦视紫红质（NaR），它在C螺旋（N112，D116和Q123）上包含独特的氨基酸残基，被称为NDQ基序。基于最近的KR2的X射线晶体结构，已通过各种方法研究了钠转运途径。然而，由于在黑暗状态下质子化的席夫碱（PRSB）区域周围的结构信息复杂，并且在KR2中结合了钠的中间体缺乏结构信息，因此钠泵的详细机理仍不清楚。在这里，我们对同位素标记的KR2 WT和定点突变蛋白（N112A，D116E，R109A和R109K）应用了全面的低温光诱导差异FTIR光谱。我们指定了PRSB在2095 cm-1处的ND拉伸振动，并阐明了与D116（PRSB的抗衡离子）的氢键相互作用。我们还在R109和D251附近分配了强氢键结合的水（2333 cm-1），发现在R109位置存在正电荷是KR2泵浦功能的先决条件。