质子转移反应在酶和转运蛋白等多种蛋白质中发挥着重要的功能作用,这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。其中一种特殊情况可以在质子化形式的海洋细菌TAT视紫红质中看到,通过光异构化视网膜,但在10-5秒内返回到原始状态。因此,光能被转化为热能而没有任何功能。相比之下,TAT视紫红质的T82D突变体在高pH下进行希夫碱的光诱导去质子化。在本文中,我们通过差分FTIR光谱报告了T82D的结构分析。在77K的光诱导差异FTIR光谱中,我们观察到T82D和野生型(WT)的HOOP振动很小,这表明平面生色团结构本身不是WTTAT视紫红质中K中间体逆转的起源。在K中间体松弛时,T82D形成以下中间体,如M,而WT的K返回到原始状态。目前的FTIR分析揭示了在形成M中间体时在T82D中从席夫碱到D82的质子转移。它伴随着从E54到希夫碱的第二次质子转移,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和席夫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中低亲和力的起源,其中E54的去质子化参与Ca2+结合。(282字)
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 intra-molecular 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 sec. 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 paper, we report the structural analysis of T82D by means of difference FTIR spectroscopy. In the light-induced difference FTIR spectra at 77 K, we observed little HOOP 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 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. Altered hydrogen-bonding network would be the origin of low affinity in T82D, where deprotonation of E54 is involved in the Ca2+ binding. (282 words).