RESONANCE RAMAN-SPECTRA OF BACTERIORHODOPSINS PRIMARY PHOTOPRODUCT - EVIDENCE FOR A DISTORTED 13-CIS RETINAL CHROMOPHORE

The resonance Raman spectrum of bacteriorhodopsin''s primary photoproduct K with a novel low-temperature spinning sample technique was obtained. Purple membrane at 77.degree. K is illuminated with spatially separated actinic (pump) and probe laser beams. The 514-nm pump beam produces a photostationary steady-state mixture of bacteriorhodopsin and K. This mixture is then rotated through the red (676 nm) probe beam, which selectively enhances the Raman scattering from K. The essential advantage of the successive pump-and-probe technique is that it prevents the fluorescence excited by the pump beam from masking the red probe Raman scattering. K exhibits strong Raman lines at 1516, 1294, 1012, 957 and 811 cm-1. The effects of C15 deuteration on K fingerprint lines correlate well with those seen in 13-cis model compounds, indicating that K has a 13-cis chromophore. The presence of unusually strong low-wave number lines at 811 and 957 cm-1, attributable to hydrogen out-of-plane wags, indicates that the protein holds the chromophore in a distorted conformation after trans .fwdarw. cis isomerization.