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Real-time spectroscopy of transition states in bacteriorhodopsin during retinal isomerization

Nature volume 414, pages 531–534 (2001)Cite this article

Abstract

Real-time investigations of the rearrangement of bonds during chemical transformations require femtosecond temporal resolution, so that the atomic vibrations within the reacting molecules can be observed. Following the development of lasers capable of emitting ultrashort laser flashes on this timescale, chemical reactions involving relatively simple molecules have been monitored in detail, revealing the transient existence of intermediate species as reactants are transformed into products1,2,3. Here we report the direct observation of nuclear motion in a complex biological system, the retinal chromophore of bacteriorhodopsin (bR568)4, as it undergoes the trans–cis photoisomerization that is fundamental to the vision process. By using visible-light pulses of less than 5 femtosecond in duration5,6, we are able to monitor changes in the vibrational spectra of the transition state and thus show that despite photoexcitation of the anti-bonding molecular orbital involved, isomerization does not occur instantly, but involves transient formation of a so-called ‘tumbling state’. Our observations thus agree with growing experimental7,8,9,10,11,12,13,14 and ab initio evidence15,16 for a three-state photoisomerization model8,9,10,17 and firmly discount the initially suggested two-state model18,19,20 for this process.

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Figure 1: Schematic potential energy curves of two-state and three-state models along the torsional angle of the C13 = C14 bond of the protonated retinal Schiff base of bR568.
Figure 2: Time-dependence of the transmittance change (ΔT/T) following sub-5-fs pulse excitation of a suspension of the light-adapted purple membrane of Halobacterium salinarum containing bR568 at room temperature.
Figure 3: Spectrogram calculated for the trace at 610 nm.
Figure 4: Fourier power spectrum of instantaneous frequencies appearing in the trace of transmittance change probed at 630 nm.
Figure 5: A mechanism of the trans–cis photoisomerization of the retinal chromophore.

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References

  1. Polanyi, J. C. & Zewail, A. H. Direct observation of the transition state. Acc. Chem. Res. 28, 119–132 (1995).

    Article  CAS  Google Scholar 

  2. Rose, T. S., Rosker, M. J. & Zewail, A. H. Femtosecond real-time observation of wave packet oscillations (resonance) in dissociation reactions. J. Chem. Phys. 88, 6672–6673 (1988).

    Article  ADS  CAS  Google Scholar 

  3. Rose, T. S., Rosker, M. J. & Zewail, A. H. Femtosecond real-time probing of reactions. IV. The reactions of alkali halides. J. Chem. Phys. 91, 7415–7436 (1989).

    Article  ADS  CAS  Google Scholar 

  4. Oesterhelt, D. & Stoeckenius, W. in Methods of Enzymology, Biomembranes Part A, Vol. 31 (eds Freischer, S. and Packer, L.) 667–678 (Academic, New York, 1974).

    Google Scholar 

  5. Shirakawa, A., Sakane, I. & Kobayashi, T. Pulse-front-matched optical parametric amplification for sub-10-fs pulse generation tunable in the visible and near infrared. Opt. Lett. 23, 1292–1294 (1998).

    Article  ADS  CAS  Google Scholar 

  6. Shirakawa, A., Sakane, I., Takasaka, M. & Kobayashi, T. Sub-5-fs visible pulse generation by pulse-front-matched noncollinear optical parametric amplification. Appl. Phys. Lett. 74, 2268–2270 (1999).

    Article  ADS  CAS  Google Scholar 

  7. Atkinson, G. H., Ujj, L. &. & Zhou, Y. Vibrational spectrum of the J-625 intermediate in the room temperature bacteriorhodopsin photocycle. J. Phys. Chem. A 104, 4130–4139 (2000).

    Article  CAS  Google Scholar 

  8. Hasson, K. C., Gai, F. & Anfinrud, P. A. The photoisomerization of retinal in bacteriorhodopsin: experimental evidence for a three-state model. Proc. Natl Acad. Sci. USA 93, 15124–15129 (1996).

    Article  ADS  CAS  Google Scholar 

  9. Haran, G. et al. Excited state dynamics of bacteriorhodopsin revealed by transient stimulated emission spectra. Chem. Phys. Lett. 261, 389–395 (1996).

    Article  ADS  CAS  Google Scholar 

  10. Gai, F., Hasson, K. C., McDonald, J. C. & Anfinrud, P. A. Chemical dynamics in proteins: the photoisomerization of retinal in bacteriorhodopsin. Science 279, 1886–1891 (1998).

    Article  ADS  CAS  Google Scholar 

  11. Humphrey, W., Lu, H., Logonov, I., Werner, H.-J. & Schulten, K. Three electronic state model of the primary phototransformation of bacteriorhodopsin. Biophys. J. 75, 1689–1699 (1998).

    Article  CAS  Google Scholar 

  12. Zhong, Q., Ruhman, S. & Ottolenghi, M. Reexamining the primary light-induced events in bacteriorhodopsin using a synthetic C13 = C14-locked chromophore. J. Am. Chem. Soc. 118, 12828–11829 (1996).

    Article  CAS  Google Scholar 

  13. Ye, T. et al. On the nature of the primary light-induced events in bacteriorhodopsin: ultrafast spectroscopy of native and C13 = C14 locked pigments. J. Phys. Chem. B 103, 5122–5130 (1999).

    Article  CAS  Google Scholar 

  14. Song, L. & El-Sayed, M. A. Primary step in bacteriorhodopsin photosynthesis: bond stretch rather than angle twist of its retinal excited-state structure. J. Am. Chem. Soc. 120, 8889–8890 (1998).

    Article  CAS  Google Scholar 

  15. Vreven, T. et al. Ab initio photoisomerization dynamics of a simple retinal chromophore model. J. Am. Chem. Soc. 119, 12687–12688 (1997).

    Article  CAS  Google Scholar 

  16. Garavelli, M., Negri, F. & Olivucci, M. Initial excited-state relaxation of the isolated 11-cis protonated Schiff base of retinal: Evidence for in-plane motion from ab initio quantum chemical simulation of the resonance Raman spectrum. J. Am. Chem. Soc. 121, 1023–1029 (1999).

    Article  CAS  Google Scholar 

  17. Du, M. & Fleming, G. R. Femtosecond time resolved fluorescence spectroscopy of bacteriorhodopsin: direct observation of excited state dynamics in the primary step of the proton pump cycle. Biophys. Chem. 48, 101–111 (1993).

    Article  CAS  Google Scholar 

  18. Peteanu, L. A., Shoenlein, R. W., Wang, Q., Mathies, R. A. & Shank, C. V. The first step in vision occurs in femtoseconds: complete blue and red spectral studies. Proc. Natl Acad. Sci. USA 90, 11762–11766 (1993).

    Article  ADS  CAS  Google Scholar 

  19. Shoenlein, R. W., Peteanu, L. A., Wang, Q., Mathies, R. A. & Shank, C. V. Femtosecond dynamics of cis-trans isomerization in a visual pigment analog: isorhodopsin. J. Phys. Chem. 97, 12087–12092 (1993).

    Article  Google Scholar 

  20. Mathies, R. A., Brito Cruz, C. H., Pollard, W. T. & Shank, C. V. Direct observation of the femtosecond excited-state cis-trans isomerization in bacteriorhodopsin. Science 240, 777–779 (1988).

    Article  ADS  CAS  Google Scholar 

  21. Atkinson, G. H., Brack, T. L., Blanchard, D. & Rumbles, G. Picosecond time-resolved resonance Raman spectroscopy of the initial trans to cis isomerization in the bacteriorhodopsin photocycle. Chem. Phys. 131, 1–15 (1989).

    Article  CAS  Google Scholar 

  22. van den Berg, R., Jang, D. J., Bitting, H. C. & El-Sayed, M. A. Subpicosecond resonance Raman spectra of the early intermediates in the photocycle of bacteriorhodopsin. Biophys. J. 58, 135–141 (1990).

    Article  CAS  Google Scholar 

  23. Doig, S. J., Reid, P. J. & Mathies, R. A. Picosecond time-resolved resonance Raman spectroscopy of bacteriorhodopsin J, K, and KL intermediates. J. Phys. Chem. 95, 6372–6379 (1991).

    Article  CAS  Google Scholar 

  24. Diller, R. et al. Femtosecond time-resolved infrared laser study of the J-K transition of bacteriorhodopsin. Chem. Phys. Lett. 241, 109–115 (1995).

    Article  ADS  CAS  Google Scholar 

  25. Pollard, W. T. et al. Theory of dynamic absorption spectroscopy of nonstationary states. 4. Application to 12-fs resonant impulsive Raman spectroscopy of bacteriorhodopsin. J. Phys. Chem. 96, 6147–6158 (1992).

    Article  CAS  Google Scholar 

  26. Bardeen, C. J., Wang, Q. & Shank, C. V. Femtosecond chirped pulse excitation of vibrational wave packets in LD690 and bacteriorhodopsin. J. Phys. Chem. A 102, 2759–2766 (1998).

    Article  CAS  Google Scholar 

  27. Eyring, G., Curry, B., Broek, A., Lugtenburg, J. & Mathies, R. Assignment and interpretation of hydrogen out-of-plane vibrations in the resonance Raman spectra of rhodopsin and bathorhodopsin. Biochemistry 21, 384–393 (1982).

    Article  CAS  Google Scholar 

  28. Myers, A. B., Harris, R. A. & Mathies, R. A. Resonance Raman excitation profiles of bacteriorhodopsin. J. Chem. Phys. 79, 603–613 (1983).

    Article  ADS  CAS  Google Scholar 

  29. Kobayashi, T., Shirakawa, A., Matsuzawa, H. & Nakanishi, H. Real-time vibrational mode-coupling associated with ultrafast geometrical relaxation in polydiacetylene induced by sub-5-fs pulses. Chem. Phys. Lett. 321, 385–393 (2000).

    Article  ADS  CAS  Google Scholar 

  30. Kobayashi, T. & Shirakawa, A. Tunable visible and near-infrared pulse generator in a 5 fs regime. Appl. Phys. B 70, S239–S246 (2000).

    Article  ADS  CAS  Google Scholar 

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Acknowledgements

We thank J. Watson and M. Murao for careful reading of the manuscript. This work was partially supported by the Research for the Future program run by the Japan Society for Promotion of Science (T.K.), the Special Coordination Funds (“Molecular Sensors for Aero-Thermodynamic Research”; H.O.) and Scientific Research (H.O.) of the Ministry of Education, Culture, Sports, Science and Technology.

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Authors and Affiliations

  1. Department of Physics, Graduate School of Science, University of Tokyo, Hongo 7-3-1, Bunkyo, 113-0033, Tokyo, Japan

    Takayoshi Kobayashi & Takashi Saito

  2. Department of Biomolecular Engineering, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, 4259, Nagatsuta, Yokohama, 226-8501, Japan

    Hiroyuki Ohtani

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Correspondence to Takayoshi Kobayashi.

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Kobayashi, T., Saito, T. & Ohtani, H. Real-time spectroscopy of transition states in bacteriorhodopsin during retinal isomerization. Nature 414, 531–534 (2001). https://doi.org/10.1038/35107042

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