Vibrational Spectrum of K-590 Containing13C14,15Retinal: Picosecond Time-Resolved Coherent Anti-Stokes Raman Spectroscopy of the Room Temperature Bacteriorhodopsin Photocycle

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The C−C stretching vibrations (1100−1400 cm-1) of the K-590 intermediate (containing a 13C14,15 retinal), formed during the room-temperature (RT) bacteriorhodopsin (BR) photocycle, are measured using picosecond time-resolved coherent anti-Stokes Raman scattering (PTR/CARS). Although time-resolved resonance Raman data have been published previously for intermediates in the room-temperature BR photocycle, these PTR/CARS data are the first time-resolved vibrational spectra from a picosecond BR intermediate at RT containing an isotopically-labeled (13C) retinal. The C14 and C15 positions are selected for isotopic labeling because motions around the C13C14 and C14−C15 bonds are thought to underlie the structural transformation from BR-570 to K-590 and, therefore, the energy storage and transduction mechanism in the RT/BR photocycle. These PTR/CARS data are recorded 50 ps after the BR photocycle is initiated with 570-nm (5 ps, fwhm) excitation and are fit to within <1 cm-1 via third-order nonlinear susceptibility (χ(3)) relationships. Comparisons of these PTR/CARS data at RT with the results from earlier resonance Raman (RR) studies of K-590 at low temperature (LT) reveal new temperature effects. Specifically, three CARS bands (1197, 1184, and 1167 cm-1) are observed from 13C14,15 K-590 in H2O samples via PTR/CARS at RT, while only two bands (1189 and 1170 cm-1) are found in LT/RR measurements from 13C14,15 K-625. Analogous temperature-dependent differences are found in data measured from 13C14,15 K-590 in D2O samples. Independently, PTR/CARS data at RT demonstrate that deuteration of the Schiff-base nitrogen causes major changes in the fingerprint region: the 1197-cm-1 band decreases to 1193 cm-1 while diminishing in intensity by half and a new band appears at 1206 cm-1. No such deuterium effect is observed in the LT/RR data from 13C14,15 K-590. The previously unrecognized sensitivity of fingerprint bands to deuteration of the Schiff-base nitrogen suggests that the C−C stretching modes are highly mixed with each other and coupled to the N−H(D) retinal rocking mode. Although the temperature and Schiff-base deuteration effects reported here had not been previously identified in LT vibrational data from K-625, an analysis of the RT/CARS data continues to support the 13-cis, 14-trans retinal structure in K-590 proposed from LT results.