Picosecond time-resolved resonance Raman spectroscopy of the initial trans to cis isomerization in the bacteriorhodopsin photocycle

The resonance Raman (RR) spectra in the fingerprint region (1100-1300 cm^-1) are reported for the initial, picosecond interval of the bacteriorhodopsin (BR) photocycle during which the J-625 and K-590 intermediates are formed. These are the first RR features assigned to J-625 while the RR bands assignable to K-590 alone are clarified with respect to previous studies. The assignment of RR features to J-625 and K-590 is based on the results of two-laser, picosecond time-resolved resonance Raman (PTR³) experiments designed to separate RR bands of K-590 from other species. The instrumental design underlying PTR³ experiments and the associated advantages for analyzing time-resolved data are described. The PTR³ data in the fingerprint region suggest that neither J-625 nor K-590 contain all-trans retinal and therefore, establish that the primary BR photocycle event involves a configurational change in the retinal chromophore. The RR fingerprint bands assignable to J-625 and K-590, however, differ from one another indicating that the two intermediates do not contain retinal in the same configurational or conformational form and that a second change in retinal structure occurs over the initial 10 ps of the photocycle. The identification of either intermediate in terms of an absolute configuration (e.g., 13-cis retinal) or conformation remains unresolved.