Krokinobacter rhodopsin 2 (KR2), a light-driven Na⁺ pump, is a dual-functional protein, pumping protons in the absence of Na⁺ when K⁺ or larger alkali metal ions are present. A specific mutation in helix A near the extracellular Na⁺ binding site, H30A, eliminates its proton pumping ability. We induced structural changes in H30A by altering the alkali metal ion bound at the extracellular binding site, and observed a strong electrostatic interaction between the Schiff base and counterion and torsion around the Schiff base as revealed by solid-state nuclear magnetic resonance (NMR) and Fourier transform infrared (FTIR) spectroscopies. The strong interaction when His30 was absent and no ion bound at the extracellular binding site disabled retinal reisomerization, as was shown with flash-photolysis, forming a small amount of only a K-like intermediate. This revealed why H30A lacks the proton pumping function. Long-distance perturbation of the binding site and Schiff base revealed that a non-transported ion binding at the extracellular site is essential for pumping.

中文翻译：

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His30和克罗迪诺视紫红质细胞外Na ⁺结合位点对席夫碱-抗衡相互作用的长距离扰动2 †

Krokinobacter rhodopsin 2（KR2），一种光驱动的Na ⁺泵，是一种双功能蛋白，当存在K ⁺或更大的碱金属离子时，在没有Na ⁺的情况下泵送质子。细胞外Na ⁺附近螺旋A的一个特定突变结合位点H30A消除了其质子泵送能力。我们通过改变结合在细胞外结合位点的碱金属离子诱导了H30A的结构变化，并观察到了席夫碱与席夫碱周围的抗衡离子和扭转之间的强静电相互作用，这由固态核磁共振（NMR）和傅立叶红外光谱（FTIR）。缺少His30且在细胞外结合位点未结合任何离子时，强烈的相互作用使视网膜再异构化失效，如快速光解法所示，仅形成少量的K型中间体。这揭示了为什么H30A缺乏质子泵送功能。结合位点和席夫碱的长距离扰动表明，胞外位点的非转运离子结合是泵浦必不可少的。