Vibrational spectrum of the K-590 intermediate in the bacteriorhodopsin photocycle at room temperature: picosecond time-resolved resonance coherent anti-Raman spectroscopy Original Research Article

The vibrational spectrum of the K-590 intermediate, thought to contribute significantly to the energy storage and transduction mechanism in the bacteriorhodopsin (BR) photocycle, is measured at room temperature using picosecond time-resolved resonance coherent anti-Stokes Raman scattering (PTR/CARS). The room-temperature BR photocycle is initiated by the 3 ps, 570 nm excitation of the ground-state species, BR-570, prepared in both H2O and D2O suspensions of BR. PTR/CARS data, recorded 50 ps after BR-570 excitation, at which time only BR-570 and K-590 are present, have an excellent S/N which provides a significantly more detailed view of the K-590 vibrational degrees of freedom than previously available. Two picosecond (6 ps FWHM) laser pulses, ω1 (633.4 nm) and ωS (675–700 nm), are used to record PTR/CARS data via electronic resonance enhancement in both BR-570 and K-590, each of which contains a distinct retinal structure (assigned as 13-rans, 15-anti, 13-cis, respectively). To obtain the vibrational spectrum of K-590 separately, the PTR/CARS spectra from the mixture of isomeric retinals is quantitatively analyzed in terms of third-order susceptibility (η(3)) relationships. PTR/CARS spectra of K-590 recorded from both H2O and D2O suspensions of BR are compared with the analogous vibrational data obtained via spontaneous resonance Raman (RR) scattering at both low (77 K) and room temperature. Analyses of these vibrational spectra identify temperature-dependent effects and changes assignable to the substitution of deuterium at the Schiff-base nitrogen not previously reported.