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Ultrafast Pump−Probe Study of the Primary Photoreaction Process in pharaonis Halorhodopsin: Halide Ion Dependence and Isomerization Dynamics

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journal contribution
posted on 09.10.2008 by Takumi Nakamura, Satoshi Takeuchi, Mikihiro Shibata, Makoto Demura, Hideki Kandori, Tahei Tahara
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 ∼30 fs time resolution. All of the temporal behaviors of the Sn ← S1 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 (S1r) and nonreactive (S1nr) S1 states. With the improved time resolution, it was revealed that the time constant of this branching process (τ1) is as short as 50 fs. The second dynamics was the isomerization process of the S1r state to generate the ground-state 13-cis form, and the time constant (τ2) 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 S1 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 S1 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 S1nr state also showed notable halide ion dependence (τ3 = 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 S1nr state.