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

Photoisomerization of an all-trans-retinal chromophore triggers ion transport in microbial ion-pumping rhodopsins. Understanding chromophore structures in the electronically excited (S₁) 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 S₁-state chromophore in Natronomonas pharaonis halorhodopsin (NpHR), a chloride ion-pumping rhodopsin, using time-resolved resonance Raman spectroscopy. The spectral patterns of the S₁-state chromophore were completely different from those of the ground-state chromophore, resulting from unique vibrational characteristics and the structure of the S₁ 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 S₁-state chromophore in NpHR, indicating a twisted polyene structure. Similar frequency shifts for the C═N/C═C and C-C stretching modes of the S₁-state chromophore in NpHR were observed in the Raman spectra of sodium ion-pumping and proton-pumping rhodopsins, suggesting that these unique features are common to the S₁ states of ion-pumping rhodopsins.