Nakamura Takumi, Takeuchi Satoshi, Shibata Mikihiro, Demura Makoto, Kandori Hideki, Tahara Tahei
Molecular Spectroscopy Laboratory, RIKEN (The Institute of Physical and Chemical Research), 2-1 Hirosawa, Wako, Japan.
J Phys Chem B. 2008 Oct 9;112(40):12795-800. doi: 10.1021/jp803282s. Epub 2008 Sep 13.
Halorhodopsin is a retinal protein that acts as a light-driven chloride pump in the Haloarchaeal cell membrane. A chloride ion is bound near the retinal chromophore, and light-induced all- trans --> 13- cis isomerization triggers the unidirectional chloride ion pump. We investigated the primary ultrafast dynamics of Natronomonas pharaonis halorhodopsin that contains Cl (-), Br (-), or I (-) ( pHR-Cl (-), pHR-Br (-), or pHR-I (-)) using ultrafast pump-probe spectroscopy with approximately 30 fs time resolution. All of the temporal behaviors of the S n <-- S 1 absorption, ground-state bleaching, K intermediate (13- cis form) absorption, and stimulated emission were observed. In agreement with previous reports, the primary process exhibited three dynamics. The first dynamics corresponds to the population branching process from the Franck-Condon (FC) region to the reactive (S 1 (r)) and nonreactive (S 1 (nr)) S 1 states. With the improved time resolution, it was revealed that the time constant of this branching process (tau 1) is as short as 50 fs. The second dynamics was the isomerization process of the S 1 (r) state to generate the ground-state 13- cis form, and the time constant (tau 2) exhibited significant halide ion dependence (1.4, 1.6, and 2.2 ps for pHR-Cl (-), pHR-Br (-), and pHR-I (-), respectively). The relative quantum yield of the isomerization, which was evaluated from the pump-probe signal after 20 ps, also showed halide ion dependence (1.00, 1.14, and 1.35 for pHR-Cl (-), pHR-Br (-), and pHR-I (-), respectively). It was revealed that the halide ion that accelerates isomerization dynamics provides the lower isomerization yield. This finding suggests that there is an activation barrier along the isomerization coordinate on the S 1 potential energy surface, meaning that the three-state model, which is now accepted for bacteriorhodopsin, is more relevant than the two-state model for the isomerization process of halorhodopsin. We concluded that, with the three-state model, the isomerization rate is controlled by the height of the activation barrier on the S 1 potential energy surface while the overall isomerization yield is determined by the branching ratios at the FC region and the conical intersection. The third dynamics attributable to the internal conversion of the S 1 (nr) state also showed notable halide ion dependence (tau 3 = 4.5, 4.6, and 6.3 ps for pHR-Cl (-), pHR-Br (-), and pHR-I (-)). This suggests that some geometrical change may be involved in the relaxation process of the S 1 (nr) state.
盐视紫红质是一种视网膜蛋白，在嗜盐古菌细胞膜中作为光驱动的氯离子泵发挥作用。氯离子结合在视网膜发色团附近，光诱导的全反式→13-顺式异构化触发单向氯离子泵。我们使用时间分辨率约为30飞秒的超快泵浦-探测光谱，研究了含有Cl(-)、Br(-)或I(-)（pHR-Cl(-)、pHR-Br(-)或pHR-I(-)）的法老盐单胞菌盐视紫红质的初级超快动力学。观察到了S n←S 1吸收、基态漂白、K中间体（13-顺式形式）吸收和受激发射的所有时间行为。与先前的报道一致，初级过程表现出三种动力学。第一种动力学对应于从弗兰克-康登（FC）区域到反应性（S 1 (r)）和非反应性（S 1 (nr)）S 1态的布居分支过程。随着时间分辨率的提高，发现该分支过程的时间常数（tau 1）短至50飞秒。第二种动力学是S 1 (r)态的异构化过程，以生成基态13-顺式形式，时间常数（tau 2）表现出显著的卤离子依赖性（pHR-Cl(-)、pHR-Br(-)和pHR-I(-)分别为1.4、1.6和2.2皮秒）。从20皮秒后的泵浦-探测信号评估的异构化相对量子产率也显示出卤离子依赖性（pHR-Cl(-)、pHR-Br(-)和pHR-I(-)分别为1.00、1.14和1.35）。结果表明，加速异构化动力学的卤离子提供了较低的异构化产率。这一发现表明，在S 1势能面上的异构化坐标上存在一个活化能垒，这意味着目前被接受的细菌视紫红质的三态模型比盐视紫红质异构化过程的二态模型更适用。我们得出结论，在三态模型中，异构化速率由S 1势能面上活化能垒的高度控制，而整体异构化产率由FC区域的分支比和锥形交叉点决定。归因于S 1 (nr)态内转换的第三种动力学也表现出显著的卤离子依赖性（pHR-Cl(-)、pHR-Br(-)和pHR-I(-)的tau 3分别为4.5、4.6和6.3皮秒）。这表明在S 1 (nr)态的弛豫过程中可能涉及一些几何变化。
