Vibrational Spectrum of the Lumi Intermediate in the Room Temperature Rhodopsin Photo-Reaction

The vibrational spectrum (650–1750 cm −1) of the lumi-rhodopsin (lumi) intermediate formed in the microsecond time regime of the room-temperature rhodopsin (Rh RT) photoreaction is measured for the first time using picosecond time-resolved coherent anti-Stokes Raman spectroscopy (PTR/CARS). The vibrational spectrum of lumi is recorded 2.5 μs after the 3-ps, 500-nm excitation of Rh RT. Complementary to Fourier transform infrared spectra recorded at Rh sample temperatures low enough to freeze lumi, these PTR/CARS results provide the first detailed view of the vibrational degrees of freedom of room-temperature lumi (lumi RT) through the identification of 21 bands. The exceptionally low intensity (compared to those observed in batho RT) of the hydrogen out-of-plane (HOOP) bands, the moderate intensity and absolute positions of C-C stretching bands, and the presence of high-intensity C C stretching bands suggest that lumi RT contains an almost planar (nontwisting), all- trans retinal geometry. Independently, the 944-cm −1 position of the most intense HOOP band implies that a resonance coupling exists between the out-of-plane retinal vibrations and at least one group among the amino acids comprising the retinal binding pocket. The formation of lumi RT, monitored via PTR/CARS spectra recorded on the nanosecond time scale, can be associated with the decay of the blue-shifted intermediate (BSI RT) formed in equilibrium with the batho RT intermediate. PTR/CARS spectra measured at a 210-ns delay contain distinct vibrational features attributable to BSI RT, which suggest that the all- trans retinal in both BSI RT and lumi RT is strongly coupled to part of the retinal binding pocket. With regard to the energy storage/transduction mechanism in Rh RT, these results support the hypothesis that during the formation of lumi RT, the majority of the photon energy absorbed by Rh RT transfers to the apoprotein opsin.