To obtain high quality time-resolved ultraviolet resonance Raman (UVRR) spectra of
transient intermediates in the bacteriorhodopsin (BR) photocycle, we developed a new
UVRR spectrometer. A home-made F=3.5 prism prefilter was used in front of a 50 cm
CCD detected spectrograph to give high throughput, wide tunability, and excellent stray
light rejection along with low dispersion. Using this system, we obtained 239.5 nm
excited time-resolved UVRR spectra of BR which revealed small but significant features
associated with the formation of the KL-intermediate at 10 ns delays. This difference
spectrum exhibits intensity decreases at 1624, 1561, 1012 and 763cm-1 due to an altered
environment of one or more Trp residues and a frequency shift of the Tyr ʋ8b band at
1602 cm-1. These signals show that the photoisomerization of retinal from all-trans to
13-cis induces significant changes in the structure and environment of aromatic residues
that line the retinal binding pocket in only 10 ns.