Resonance Raman structural evidence that the cis-to-trans isomerization inrhodopsin occurs in femtoseconds

Picosecond time-resolved resonance Raman spectroscopy is used to probe the structural changes of rhodopsin's retinal chromophore as the cis-to-trans i somerization reaction occurs that initiates vision. Room-temperature resona nce Raman spectra of rhodopsin's photoproduct with time delays from -0.7 to 20.8 ps are measured using 2.2 ps, 480 nm pump and 1.5 ps, 600 nm probe pu lses. Hydrogen-out-of-plane (HOOP) modes at 852, 871, and 919 cm(-1), finge rprint peaks at 1272, 1236, 1211, and 1166 cm(-1), and a broad red-shifted ethylenic band at 1530 cm(-1) are present at the earliest positive pump-pro be time delay of 0.8 ps, indicating that the chromophore is already in a st rained, all-trans configuration. Kinetic analyses of both the HOOP and ethy lenic regions of the photoproduct spectra reveal that these features grow i n with fast (similar to 200 fs) and slow (similar to 2-3 ps) components. Th ese data provide the first structural evidence that photorhodopsin has a th ermally unrelaxed, torsionally strained all-trans chromophore within simila r to1 ps, and possibly within 200 fs, of photon absorption. Following this ultrafast product formation, the all trans chromophore cools and conformati onally relaxes within a few picoseconds to form bathorhodopsin. This coolin g process is revealed as an ethylenic frequency blueshift of 6 cm(-1) (tau similar to 3.5 ps) as well as an ethylenic width narrowing (tau similar to 2 ps). The ultrafast production of photorhodopsin is likely accompanied by an impulsively driven, localized protein response. More delocalized protein modes are unable to relax on this ultrafast time scale enabling the chromo phore-protein complex to store the large amounts of photon energy (30-35 kc al/mol) that are subsequently used to drive activating protein conformation al changes.