1. 1 The Initial Photochemical Process of Retinal in Bacteriorhodopsin MIYASAKA Lab. Tetsuro KATAYAMA

2. 2 Contents (1)“Probing the ultrafast charge translocation of photoexcited retinal in bacteriorhodopsin” (1) S.Schenkl, F. van Mourik, G. vander Zwan, S. Haacke, M. Chergui. Science, 2005, 309, 917 Photoisomerization ；光異性化反応 Introduction ・ Bacteriorhodopsin ・ Photoisomerization Experimental ・ Pump-probe spectroscopy Result and Discussion ・ Probing Tryptophan residues Summary

3. 3 Organisms which can convert light energy into chemical energy Halobacterium halobium Purple membrane consists of bacteriorhodopsin Halobacteria in the Pink Salt Lakes http://www.striderusa.com/salt.html http://www.unigiessen.de/~gf1265/GROUPS/KLUG/extrem_halophile.html

4. 4 Bacteriorhodopsin resembles photosynthesis and rhodopsin Bacterio- rhodopsin PhotosynthesisRhodopsin FunctionLight to energy Light to signal Initial process Isomerization of retinal Charge separation and electron transfer Isomerization of retinal Existing place Plasma membrane ChloroplastRetina Plasma membrane ; 細胞膜 Chloroplast ; 葉緑体 Retina ; 網膜

5. 5 Mechanism of bacteriorhodopsin’s photoisomerization is different from that of rhodopsin F. Gai et al., Science, 1998, 279, 1886 Schiff base Rhodopsin D.W. McCamant et al., Science, 2005, 310, 1006 Bacteriorhodopsin

6. 6 Cycle of isomerization of retinal F. Garczarek et al., Nature, 2006, 439, 109 ～ 1μs ～ 100μs >ms Process of isomerization to K BR J K >ms ～ 500fs ～ 3ps S. O. Smith et al., Proc. Natl. Acad. Sci. USA, 1984, 81, 2055

7. 7 H. Luecke et al., Science, 1999, 286, 255 Cytosol Extracellular space Extracellular space ; Cytosol ; 細胞胞質ゾル 細胞外側 Bacteriorhodopsin is a part of solar energy transducer (1)Deprotonation of the Schiff base, protonation of Asp 85 (2)proton release to the extracellular surface (3)reprotonation of the Schiff base, deprotonation of Asp 96 (4)reprotonation of Asp 96 (5)deprotonation of Asp 85 reprotonation of the proton release site.

8. 8 Time-dependence of the transient absorption can observed by pump-probe spectroscopy Probe Pump Detector Delay Time Sample τ

9. 9 What kinds of signals are observed ? 0 t Photoinduced Absorption Energy Equilibrium Nuclear Position Excited state Ground state Equilibrium Nuclear Position ： 平衡核間位置 Photoinduced Absorption ： 光誘起吸収 Photo-bleaching ： ブリーチング Stimulated Emission ： 誘導放出 0 t A ⊿ Photo-bleaching Stimulated Emission 0 t A ⊿

10. 10 Absorption spectrum of bacteriorhodopsin consists of Trp and retinal absorbance 560nm Retinal absorbance 265 ～ 280nm Tryptophan absorbance

11. 11 Transient absorption spectra of retinal F. Gai et al., Science, 1998, 279, 1886 At 0.316ps At 31.6ps Absorbance spectrum Photoisomerization was over After 31.6ps Excited-state absorption and stimulated emission disappeared Bleach signal and Photoproduct absorption remained 460nm 560nm 630nm 910nm

12. 12 Initial process of photoisomerization The first event H→I occurs within 50-100fs The second event occurs within 100-200fs. The third event occurs within 200-600fs The fourth event occurs within 700fs-3 ps T. Kobayash.et al., Nature, 2001, 414, 531 J

13. 13 Trp residues close to retinal Transition dipole of retinal and Trp 86 are parallel

14. 14 What do components of signal indicate ? Decay component was fitted to previous temporal transient absorption (420fs=relaxation to J, 3.5ps= relaxation to K) A constant,weak bleach signal is observed at the longest delay times -1 0 1 2 3 10 Delay (ps)

15. 15 What is rise component ? Cross-correlation (FWHM=85fs) Temporal derivative bleach transient (FWHM=150fs) Rise time was observed

16. 16 Exiciton-Coupling model L a,b S1S1 SnSn X 1, X 2, X 3 ; Linear coupling of L a,b － S n XX 1, XX 2 ; Linear coupling of L a,b, － S 1

17. 17 Calculation of the effect of retinal dipole moment change Bleach signal increase Dipole moment of retinal increase  A* shifts red  A doesn’t change

18. 18 Conclusion Charge translocation occurs The isomerization takes place after charge translocation. K J

19. 19 Summary Time scale of charge translocation and its interplay with the initial twisting and subsequent isomerization were confirmed.

20. 20 Landau-zener equation