TIME-RESOLVED RESONANCE RAMAN SPECTROSCOPY OF THE BACTERIORHODOPSIN PHOTOCYCLE ON THE PICOSECOND AND NANOSECOND TIME SCALES

Abstract— Resonance Raman spectra of the picosecond bacteriorhodopsin intermediate(s) have been obtained by microbeam, flow and subtraction techniques using a synchronously pumped, cavity-dumped dye laser. Nanosecond spectra also were measured with this laser by cavity dumping without mode-locking. The picosecond spectra in the fingerprint region, which is sensitive to the configuration of the retinal chromophore, differ from spectra of the parent bR₅₇₀ but could be correlated to the spectrum of bR^DA₅₅₀ , a “13-cis” species which has been determined from spectra of bR₅₇₀ and bR^DA₅₆₀. The picosecond transient and bR^DA₅₅₀ also are similar in the 950–1050 cm^-1“deuteration fingerprint” region when the medium is changed from H₂O to D₂O. These results suggest that trans—cis isomerization occurs during the 40-ps pulse duration. The shift relative to the parent bR₅₇₀ in the ethylenic stretch region suggests that the picosecond and nanosecond transients absorb at wavelengths longer than 570 nm. The C band at 1646 cm^-1 is found to shift or to broaden upon photolysis in the picosecond time scale. This might suggest a change in the electronic structure of the group and its environment on the picosecond time domain. The nanosecond spectra obtained in this work (with 15-ns pulses) are similar to the spectra previously observed on the 100-ns time scale but are slightly different from the picosecond spectrum. These data suggest that more than one transient species appears on the picosecond-to-nanosecond time scale. The temporal evolution of Raman bands in the fingerprint as well as the low energy (950–1050 cm^-1) region and its implications are discussed.