The vibrational spectra of the batho and lumi intermediates in the room‐temperature rhodopsin photo‐reaction are measured by picosecond time‐resolved coherent anti‐Stokes Raman spectroscopy (PTR/CARS). The instrumental principles underlying PTR/ CARS together with the advantages of PTR/CARS applications in protein reactions are discussed.

The structural changes in the retinal chromophore that underlie the initial picosecond processes in the room temperature rhodopsin (RhRT) photo-reaction (i.e., photoRT and bathoRT intermediates) are examined through the vibrational spectra of four artificial Rh pigments (new vibrational spectra from three artificial Rh pigments are presented here). Each of these Rh pigments contains a retinal in which isomerization around the C11C12 bond, thought to be the primary reaction coordinate in the RhRT photo-reaction, is blocked. Specifically, vibrational spectra from the ground electronic states of Rh7.10 (containing a 7-membered carbon ring spanning the C10–C11C12–C13 bonds in the retinal), Rh7.10/8D (Rh7.10 containing a deuterium at C8), Rh7.10/ND (Rh7.10 containing a deuterium at the Schiff-base nitrogen) and Rh8.10 (containing an 8-membered carbon ring spanning the C10–C11C12–C13 bonds in the retinal) are recorded by picosecond resonance coherent anti-Stokes Raman spectroscopy (PR/CARS). These PR/CARS data are analyzed with the aid of vibrational mode, assignments originating with native Rh used to identify the structural motions that can be associated with specific vibrational features. These PR/CARS assignments provide the spectroscopic information required to interpret the structural changes in the retinal chromophore, observed via picosecond time-resolved CARS measurements (reported elsewhere), following optical excitation of these same artificial Rh pigments. The relationships between structural changes in these artificial Rh pigments and those occurring in native Rh are also discussed.

The vibrational degrees of freedom of the only photophysical intermediate formed during the photoreaction of an artificial rhodopsin (Rh) containing a retinal with a seven-membered ring blocking 11-cis isomerization (Rh7.10) is measured via picosecond time-resolved coherent anti-Stokes Raman spectroscopy (PTR/CARS). PTR/CARS spectra are recorded with time delays ranging from 0 (8-ps cross correlation time) to 50 ps following the 3-ps, 500-nm excitation of Rh7.10. For time delays between 0 and 15 ps, an intermediate (P7.10) with a vibrational structure distinct from that of the ground electronic state Rh7.10 is observed. Although the formation time of P7.10 cannot be resolved from these PTR/CARS data, it is estimated to be ∼1 ps (i.e., slower than the 200-fs process proposed for native Rh). P7.10 completely reforms Rh7.10 with a decay time estimated to be ∼5−6 ps from the increasing intensities (via amplitudes from third-order, nonlinear susceptibility (χ(3)) analysis) in three major PTR/CARS features (at ...

The vibrational spectrum (650–1750cm−1) of the lumi-rhodopsin (lumi) intermediate formed in the microsecond time regime of the room-temperature rhodopsin (RhRT) photoreaction is measured for the first time using picosecond time-resolved coherent anti-Stokes Raman spectroscopy (PTR/CARS). The vibrational spectrum of lumi is recorded 2.5μs after the 3-ps, 500-nm excitation of RhRT. Complementary to Fourier transform infrared spectra recorded at Rh sample temperatures low enough to freeze lumi, these PTR/CARS results provide the first detailed view of the vibrational degrees of freedom of room-temperature lumi (lumiRT) through the identification of 21 bands. The exceptionally low intensity (compared to those observed in bathoRT) of the hydrogen out-of-plane (HOOP) bands, the moderate intensity and absolute positions of C-C stretching bands, and the presence of high-intensity CC stretching bands suggest that lumiRT contains an almost planar (nontwisting), all-trans retinal geometry. Independently, the 944-cm−1 position of the most intense HOOP band implies that a resonance coupling exists between the out-of-plane retinal vibrations and at least one group among the amino acids comprising the retinal binding pocket. The formation of lumiRT, monitored via PTR/CARS spectra recorded on the nanosecond time scale, can be associated with the decay of the blue-shifted intermediate (BSIRT) formed in equilibrium with the bathoRT intermediate. PTR/CARS spectra measured at a 210-ns delay contain distinct vibrational features attributable to BSIRT, which suggest that the all-trans retinal in both BSIRT and lumiRT is strongly coupled to part of the retinal binding pocket. With regard to the energy storage/transduction mechanism in RhRT, these results support the hypothesis that during the formation of lumiRT, the majority of the photon energy absorbed by RhRT transfers to the apoprotein opsin.

Picosecond time-resolved coherent anti-Stokes Raman spectroscopy (PTR/CARS) is used to generate high signal to noise (S/N) vibrational spectra of bathorhodopsin (batho) formed in the photoreaction of room-temperature rhodopsin (Rh-RT). These PTR/CARS spectra of batho(RT), measured as a function of only the time following 3-ps (full width at half maximum), 500-nm excitation of Rh-RT, demonstrate that the vibrational structure of batho(RT) in the 700-1700-cm(-1) region is distinct from that of Rh-RT and remains unchanged over at least the 10 ps [8 ps cross correlation time (CCT)] to 100 ns interval of the Rh-RT photoreaction. Given the experimental difficulties associated with the irreversibility of the Rh-RT photoreaction, these are the first time-resolved vibrational spectra of batho(RT) over the full 700-1700-cm(-1) region to be reported. The PTR/CARS spectra taken after 100 ns contain vibrational features other than those assignable to either Rh-RT or batho(RT) (potentially assignable to the blue-shifted intermediate, BSI). Excellent agreement is found between the major features of the Rh-RT and batho(RT) vibrational spectra measured via PTR/CARS and earlier resonance Raman (RR) spectra taken at low temperatures (LT) selected to thermally stabilize (freeze) bathe. Comparisons of the C=C stretching mode region reveal a 12-cm(-1) shift upon batho(RT) formation (PTR/CARS data), which agrees well with the 13-cm(-1) shift found for batho trapped at LT (RR data), These vibrational frequency changes also correlate well with the corresponding 38-nm (LT) and 31-nm (RT) shifts observed in the absorption maxima upon the formation of baths, thereby supporting an inverse relationship between C=C frequencies and absorption maxima proposed for retinal proteins, Comparisons of the C=C stretching frequencies at LT and RT reveal a temperature dependence characterized by red shifts of 4 cm(-1) in Rh and 3 cm(-1) in bathe, the direction of which is opposite to the blue shifts observed in the visible absorption maxima of Rh (7 nm) and bathe (14 nm). This latter observation suggests a stronger interaction of the protein (likely with the counterion Glu-113) with the all-trans-retinal in the batho(RT) structure than in the corresponding static batho(LT) structure.

Time-resolved vibrational spectra are used to elucidate the structural changes in the retinal chromophore within the K-590 intermediate that precedes the formation of the L-550 intermediate in the room-temperature (RT) bacteriorhodopsin (BR) photocycle. Measured by picosecond time-resolved coherent anti-Stokes Raman scattering (PTR/CARS), these vibrational data are recorded within the 750 cm-1 to 1720 cm-1 spectral region and with time delays of 50-260 ns after the RT/BR photocycle is optically initiated by pulsed (< 3 ps, 1.75 nJ) excitation. Although K-590 remains structurally unchanged throughout the 50-ps to 1-ns time interval, distinct structural changes do appear over the 1-ns to 260-ns period. Specifically, comparisons of the 50-ps PTR/CARS spectra with those recorded with time delays of 1 ns to 260 ns reveal 1) three types of changes in the hydrogen-out-of-plane (HOOP) region: the appearance of a strong, new feature at 984 cm-1; intensity decreases for the bands at 957 cm-1, 952 cm-1, and 939 cm-1; and small changes intensity and/or frequency of bands at 855 cm-1 and 805 cm-1; and 2) two types of changes in the C-C stretching region: the intensity increase in the band at 1196 cm-1 and small intensity changes and/or frequency shifts for bands at 1300 cm-1 and 1362 cm-1. No changes are observed in the C = C stretching region, and no bands assignable to the Schiff base stretching mode (C = NH+) mode are found in any of the PTR/CARS spectra assignable to K-590. These PTR/CARS data are used, together with vibrational mode assignments derived from previous work, to characterize the retinal structural changes in K-590 as it evolves from its 3.5-ps formation (ps/K-590) through the nanosecond time regime (ns/K-590) that precedes the formation of L-550. The PTR/CARS data suggest that changes in the torsional modes near the C14-C15 = N bonds are directly associated with the appearance of ns/K-590, and perhaps with the KL intermediate proposed in earlier studies. These vibrational data can be primarily interpreted in terms of the degree of twisting of the C14-C15 retinal bond. Such twisting may be accompanied by changes in the adjacent protein. Other smaller, but nonetheless clear, spectral changes indicate that alterations along the retinal polyene chain also occur. The changes in the retinal structure are preliminary to the deprotonation of the Schiff base nitrogen during the formation of M-412. The time constant for the ps/ns K-590 transformation is estimated from the amplitude change of four vibrational bands in the HOOP region to be 40-70 ns.

The C-C stretching vibrations (1100-1400 cm(-1)) of the K-590 intermediate (containing a C-13(14),(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 (C-13) retinal. The C-14 and C-15 positions are selected for isotopic labeling because motions around the C-13=C-14 and C-14-C-15 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 (chi((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 C-13(14,15) 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 C-13(14,15) K-625. Analogous temperature-dependent differences are found in data measured from C-13(14,15) 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 C-13(14,15) 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.

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.