Takumi Nakamura

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

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.

Fluorescence Up-Conversion Study of Excitation Energy Transport Dynamics in Oligothiophene-Fullerene Linked Dyads

Photoinduced excitation energy transport dynamics in oligothiophene-fullerene linked dyads, nT-C60 (n = 4, 8, and 12), have been investigated by femtosecond fluorescence up-conversion. In 8T-C60 and 12T-C60, each time profile of the fluorescence due to the 1nT* moiety consists of two components. The sub-picosecond component and a few picosecond components were experimentally evaluated depending on the lengths of oligothiophenes (n =8 and 12) and on the analyzing wavelength of the fluorescence. However, the time trace of the fluorescence due to 14T*-C60 decayed with a single short component in approximately 300 fs due to direct excited energy transfer (EET) from the 14T* moiety to the C60 moiety. On the basis of the kinetic models considering the short and long locally pi-conjugative thiophene segments in 8T-C60 and 12T-C60, the rate parameters of the elemental processes were evaluated. Sub-picosecond time constants of nT-C60 were found to be EET from the thiophene segment vicinal to the C60 moiety and intrachain energy transfer. Slower picosecond dynamics mainly corresponds to EET from the thiophene segments apart from the C60 moiety.

Femtosecond fluorescence study of the reaction pathways and nature of the reactive S1 state of cis-stilbene.

We report a femtosecond time-resolved fluorescence study of cis-stilbene, a prototypical molecule showing ultrafast olefinic photoisomerization and photocyclization. The time-resolved fluorescence signals were measured in a nonpolar solvent over a wide ultraviolet-visible region with excitation at 270 nm. The time-resolved fluorescence traces exhibit non-single exponential decays which are well fit with bi-exponential functions with time constants of τ(A) = 0.23 ps and τ(B) = 1.2 ps, and they are associated with the fluorescence emitted from different regions of the S(1) potential energy surface (PES) in the course of the structural change. Quantitative analysis revealed that the two fluorescent components exhibit similar intrinsic time-resolved spectra extending from 320 nm to 700 nm with the (fluorescence) oscillator strength of f(A) = 0.32 and f(B) = 0.21, respectively. It was concluded that the first component is assignable to the fluorescence from the untwisted S(1) PES region where the molecule reaches immediately after the initial elongation of the central C[double bond, length as m-dash]C bond, while the second component is the fluorescence from the substantially twisted region around a shallow S(1) potential minimum. The quantitative analysis of the femtosecond fluorescence data clearly showed that the whole isomerization process proceeds in the one-photon allowed S(1) state, thereby resolving a recent controversy in quantum chemical calculations about the reactive S(1) state. In addition, the evaluated oscillator strengths suggest that the population branching into the isomerization/cyclization pathways occurs in a very early stage when the S(1) molecule still retains a planar Ph-C[double bond, length as m-dash]C-Ph skeletal structure. On the basis of the results obtained, we discuss the dynamics and mechanism of the isomerization/cyclization reactions of cis-stilbene, as well as the electronic structure of the reaction precursor.

Properties of the Anion-Binding Site of pharaonis Halorhodopsin Studied by Ultrafast Pump−Probe Spectroscopy and Low-Temperature FTIR Spectroscopy

Halorhodopsin (HR) is a light-driven chloride pump. Cl(-) is bound in the Schiff base region of the retinal chromophore, and unidirectional Cl(-) transport is probably enforced by the specific hydrogen-bonding interaction with the protonated Schiff base and internal water molecules. It is known that HR from Natronobacterium pharaonis (pHR) also pumps NO(3)(-) with similar efficiency, suggesting that NO(3)(-) binds to the Cl(-)-binding site. In the present study, we investigated the properties of the anion-binding site by means of ultrafast pump-probe spectroscopy and low-temperature FTIR spectroscopy. The obtained data were surprisingly similar between pHR-NO(3)(-) and pHR-Cl(-), even though the shapes and sizes of the two anions are quite different. Femtosecond pump-probe spectroscopy showed very similar excited-state dynamics between pHR-NO(3)(-) and pHR-Cl(-). Low-temperature FTIR spectroscopy of unlabeled and [zeta-(15)N]Lys-labeled pHR revealed almost identical hydrogen-bonding strengths of the protonated retinal Schiff base between pHR-NO(3)(-) and pHR-Cl(-), which is similarly strengthened after retinal isomerization. There were spectral variations for water stretching vibrations between pHR-NO(3)(-) and pHR-Cl(-), suggesting that the water molecules hydrate each anion. Nevertheless, the overall spectral features were similar for the two species. These observations strongly suggest that the anion-binding site has a flexible structure and that the interaction between retinal and the anions is weak, despite the presence of an electrostatic interaction. Such a flexible hydrogen-bonding network in the Schiff base region in HR appears to be in remarkable contrast to that in light-driven proton-pumping proteins.