Sub-5-fs real-time spectroscopy of transition states in bacteriorhodopsin during retinal isomerization

By using a sub-5-fs visible laser pulse, we have made the first observation of the vibrational spectra of the transition state during trans-cis isomerization in the retinal chromophore of bacteriorhodopsin (bR(S68)). No instant isomerization of the retinal occurs in spite of electron promotion from the bonding pi-orbital to the anti-bonding pi*-orbital. The difference between the in-plane and out-of-plane vibrational frequencies (about 1150-1250 and 900-1000 cm(-1), respectively) is reduced during the first time period. The vibrational spectra after this period became very broad and weak and are ascribed to a "silent state." The silent state lasts for 700-900 fs until the chromophore isomerizes to the cis-C-13=C-14 conformation. The frequency of the C = C stretching mode was modulated by the torsion mode of the C-13=C-14 double bond with a period of 200 fs. The modulation was clearly observed for four to five periods. Using the empirical equation for the relation between bond length and stretching frequency, we determined the transitional C=C bond length with about 0.01 angstrom accuracy during the torsion motion around the double bond with 1-fs time resolution.