Proton transfer reactions play important functional roles in many proteins, such as enzymes and transporters, which is also the case in rhodopsins. In fact, functional expression of rhodopsins accompanies intramolecular proton transfer reactions in many cases. One of the exceptional cases can be seen in the protonated form of marine bacterial TAT rhodopsin, which isomerizes the retinal by light but returns to the original state within 10−5 s. Thus, light energy is converted into heat without any function. In contrast, the T82D mutant of TAT rhodopsin conducts the light-induced deprotonation of the Schiff base at high pH. In this article, we report the structural analysis of T82D by means of difference Fourier transform infrared (FTIR) spectroscopy. In the light-induced difference FTIR spectra at 77 K, we observed little hydrogen out-of-plane vibrations for T82D as well as the wild-type (WT), suggesting that the planar chromophore structure itself is not the origin of the reversion from the K intermediate in WT TAT rhodopsin. Upon relaxation of the K intermediate, T82D forms the following intermediate, such as M, whereas K of WT returns to the original state. Present FTIR analysis revealed the proton transfer from the Schiff base to D82 in T82D upon formation of the M intermediate. It is accompanied by the second proton transfer from E54 to the Schiff base, forming the N intermediate, particularly in membranes. The equilibrium between the M and N intermediates corresponds to the protonation equilibrium between E54 and the Schiff base. We also found that Ca2+ binding takes place in T82D as well as WT but with 6 times lower affinity. An altered hydrogen-bonding network would be the origin of low affinity in T82D, where deprotonation of E54 is involved in the Ca2+ binding.

中文翻译：

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光诱导的质子转移和 Ca2+ 结合在 TAT 视紫红质 T82D 突变体中的 FTIR 研究


质子转移反应在许多蛋白质中起着重要的功能作用，例如酶和转运蛋白，在视紫红质中也是如此。事实上，在许多情况下，视紫红质的功能表达伴随着分子内质子转移反应。其中一种例外情况可以在海洋细菌 TAT 视紫红质的质子化形式中看到，它通过光异构化视网膜，但在 10-5 秒内恢复到原始状态。因此，光能在没有任何功能的情况下转化为热能。相反，TAT 视紫红质的 T82D 突变体在高 pH 值下传导光诱导的 Schiff 碱去质子化。在本文中，我们报道了通过差分傅里叶变换红外 （FTIR） 光谱对 T82D 进行的结构分析。在 77 K 的光诱导差分 FTIR 光谱中，我们观察到 T82D 和野生型 （WT） 的氢平面外振动很小，这表明平面发色团结构本身并不是 WT TAT 视紫红质中 K 中间体回归的来源。在 K 中间体松弛后，T82D 形成以下中间体，例如 M，而 WT 的 K 恢复到原始状态。目前的 FTIR 分析显示，在 M 中间体形成后，T82D 中的质子从 Schiff 碱转移到 D82。它伴随着从 E54 到席夫碱的第二个质子转移，形成 N 中间体，特别是在膜中。M 和 N 中间体之间的平衡对应于 E54 和 Schiff 碱之间的质子化平衡。我们还发现 Ca2+ 结合发生在 T82D 和 WT 中，但亲和力低 6 倍。改变的氢键网络将是 T82D 中低亲和力的来源，其中 E54 的去质子化参与 Ca2+ 结合。