PRESTO, Japan Science and Technology Agency , 4-1-8 Honcho Kawaguchi , Saitama 332-0012 , Japan.
Graduate School of Bioscience and Biotechnology , Tokyo Institute of Technology , Yokohama 226-8501 , Japan.
J Phys Chem B. 2018 May 10;122(18):4784-4792. doi: 10.1021/acs.jpcb.8b01934. Epub 2018 Apr 30.
KR2 is the first light-driven Na-pumping rhodopsin discovered. It was reported that the photoexcitation of KR2 generates multiple S states, i.e., "reactive" and "nonreactive" S states at physiological pH, but their origin remained unclear. In this study, we examined the S state dynamics of KR2 using femtosecond time-resolved absorption spectroscopy at different pH's in the range from 4 to 11. It was found that the reactive S state is predominantly formed at pH >9, but its population drastically decreases with decreasing pH while the population of the nonreactive S state(s) increases. The pH dependence of the relative population of the reactive S state correlates very well with the pH titration curve of Asp116, which is the counterion of the protonated retinal Schiff base (PRSB) in KR2. This strongly indicates that the deprotonation/protonation of Asp116 is directly related to the generation of the multiple S states in KR2. The quantitative analysis of the time-resolved absorption data led us to conclude that the reactive and nonreactive S states of KR2 originate from KR2 proteins having a hydrogen bond between Asp116 and PRSB or not, respectively. In other words, it is the ground-state inhomogeneity that is the origin of the coexistence of the reactive and nonreactive S states in KR2. So far, the generation of multiple S states having a different photoreactivity of rhodopsins has been mainly explained with the branching of the relaxation pathway in the Franck-Condon region in the S state. The present study shows that the structural inhomogeneity in the ground state, in particular that of the hydrogen-bond network, is the more plausible origin of the reactive and nonreactive S states which have been widely observed for various rhodopsins.
KR2 是首个被发现的光驱动的 Na 泵浦视紫红质。据报道，KR2 的光激发会产生多个 S 态，即在生理 pH 值下产生“反应性”和“非反应性”S 态，但它们的起源仍不清楚。在这项研究中，我们使用飞秒时间分辨吸收光谱法在 pH 值为 4 到 11 的范围内研究了 KR2 的 S 态动力学。结果发现，反应性 S 态主要在 pH>9 时形成，但随着 pH 值的降低，其种群数量急剧减少，而非反应性 S 态的种群数量增加。反应性 S 态相对种群的 pH 值依赖性与 Asp116 的 pH 滴定曲线非常吻合，Asp116 是 KR2 中质子化视黄醛 Schiff 碱（PRSB）的反离子。这强烈表明 Asp116 的去质子化/质子化与 KR2 中多个 S 态的产生直接相关。对时间分辨吸收数据的定量分析使我们得出结论，KR2 的反应性和非反应性 S 态分别源自 KR2 蛋白中 Asp116 和 PRSB 之间是否存在氢键。换句话说，正是基态不均匀性导致了 KR2 中反应性和非反应性 S 态的共存。到目前为止，多种具有不同光反应性的视紫红质 S 态的产生主要通过 S 态 Franck-Condon 区域中弛豫途径的分支来解释。本研究表明，基态中的结构不均匀性，特别是氢键网络的结构不均匀性，是各种视紫红质广泛观察到的反应性和非反应性 S 态的更合理起源。
