Resonance Raman Structural Evidence that the Cis-to-Trans Isomerization in Rhodopsin Occurs in Femtoseconds

Publisher Website
Google Scholar
Abstract
Picosecond time-resolved resonance Raman spectroscopy is used to probe the structural changes of rhodopsin's retinal chromophore as the cis-to-trans isomerization reaction occurs that initiates vision. Room-temperature resonance 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 pulses. Hydrogen-out-of-plane (HOOP) modes at 852, 871, and 919 cm-1, fingerprint 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−probe time delay of 0.8 ps, indicating that the chromophore is already in a strained, all-trans configuration. Kinetic analyses of both the HOOP and ethylenic regions of the photoproduct spectra reveal that these features grow in with fast (∼ 200 fs) and slow (∼ 2−3 ps) components. These data provide the first structural evidence that photorhodopsin has a thermally unrelaxed, torsionally strained all-trans chromophore within ∼1 ps, and possibly within 200 fs, of photon absorption. Following this ultrafast product formation, the all-trans chromophore cools and conformationally relaxes within a few picoseconds to form bathorhodopsin. This cooling process is revealed as an ethylenic frequency blue-shift of 6 cm-1 (τ ∼ 3.5 ps) as well as an ethylenic width narrowing (τ ∼ 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 chromophore-protein complex to store the large amounts of photon energy (30−35 kcal/mol) that are subsequently used to drive activating protein conformational changes.