Vibrational Analysis of Coherent Anti-Stokes Resonance Raman Spectra from Bacteriorhodopsin Containing Isotopically Substituted Retinal Chromophores

Abstract

Vibrational spectra recorded by coherent anti-Stokes resonance Raman scattering (CARS) from bacteriorhodopsin (BR) samples containing isotopically substituted (2H and 13C) retinal chromophores were measured using high repetition rate, low-power, picosecond pulsed excitation (λ1=580 nm and λs=640±3 nm). These picosecond resonance CARS (PR/CARS) data were analyzed via third-order susceptibility relationships [χ(3)] to obtain band origins, bandwidths, relative intensities, and electronic phase factors assignable to all significant vibrational Raman features in the 1490–1700 cm−1 wavenumber region (the ethylenic stretching and C=N–H rocking or Schiff base modes). Isotopic substitution selectively places 2H at C15, 13C singly at the C10 position and at the C14 position, and 13C simultaneously in positions of C14 and C15. Each isotopic BR sample was examined not only in H2O, but also in D2O, which places a 2H at the Schiff base nitrogen of the retinal. In addition, PR/CARS data were recorded from each isotopic BR sample following either light adaptation [i.e. the BR sample contained a single retinal isomer (all-trans, 15-anti or BR-570)] or dark adaptation [i.e. the BR sample contained a mixture of comparable amounts of retinal isomers (BR-570 and 13-cis, 15-syn or BR-548)]. Excellent agreement was found between the vibrational features observed by PR/CARS and those obtained from spontaneous resonance Raman measurements from the same isotopically substituted BR pigments. Several new vibrational features were also found from the PR/CARS data. Vibrational Raman data from three of the isotopic BR samples in D2O are reported for the first time.