Unique Vibrational Characteristics and Structures of the Photoexcited Retinal Chromophore in Ion-Pumping Rhodopsins.

Photoisomerization of an all--retinal chromophore triggers ion transport in microbial ion-pumping rhodopsins. Understanding chromophore structures in the electronically excited (S) state provides insights into the structural evolution on the potential energy surface of the photoexcited state. In this study, we examined the structure of the S-state chromophore in halorhodopsin (HR), a chloride ion-pumping rhodopsin, using time-resolved resonance Raman spectroscopy. The spectral patterns of the S-state chromophore were completely different from those of the ground-state chromophore, resulting from unique vibrational characteristics and the structure of the S state. Mode assignments were based on a combination of deuteration shifts of the Raman bands and hybrid quantum mechanics-molecular mechanics calculations. The present observations suggest a weakened bond alternation in the π conjugation system. A strong hydrogen-out-of-plane bending band was observed in the Raman spectra of the S-state chromophore in HR, indicating a twisted polyene structure. Similar frequency shifts for the C═N/C═C and C-C stretching modes of the S-state chromophore in HR were observed in the Raman spectra of sodium ion-pumping and proton-pumping rhodopsins, suggesting that these unique features are common to the S states of ion-pumping rhodopsins.