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 955, 1235, and 1551 cm-1). The structural changes occurring as P7.10 is formed can be derived from vibrational mode assignments in the PTR/CARS spectra such as the 955-cm-1 band assigned as the HC11C12H hydrogen-out-of-plane (HOOP) mode (shifts to 946 cm-1, increases intensity, and broadens its width in P7.10) and the 1551-cm-1 band assigned as the CC stretching mode (shifts to 1546 cm-1 in P7.10). These PTR/CARS data show that incorporation of an 11-ene, seven-membered ring into the retinal chromophore permits some flexibility for torsional motion around the C11C12 bond and within the seven-membered ring, but does not allow considerable out-of-plane motion near the Schiff base or the β-ionone ring. The χ(3) analysis of these PTR/CARS data demonstrates that a series of structurally-distinct retinals having slightly different C11C12 torsional, out-of-plane motion appear (collectively P7.10). The amplitudes and shifts in frequency of the PTR/CARS vibrational features change on a time scale comparable to that of vibrational relaxation in native Rh. The structural variations observed within P7.10 are located at/near the C11C12 bond (reactive in native RhRT, but nonreactive in R7.10) and the degree of π-electron energy delocalization throughout the retinal changes. This latter phenomenon is reflected in the different electronic phase factor (ϑ) found in the χ(3) analysis of the PTR/CARS data. The relationship(s) of the P7.10 structure to that of the C11C12 reaction coordinates in native Rh is discussed.
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 (RhRT). These PTR/CARS spectra of bathoRT, measured as a function of only the time following 3-ps (full width at half maximum), 500-nm excitation of RhRT, demonstrate that the vibrational structure of bathoRT in the 700−1700-cm-1 region is distinct from that of RhRT and remains unchanged over at least the 10 ps [8 ps cross correlation time (CCT)] to 100 ns interval of the RhRT photoreaction. Given the experimental difficulties associated with the irreversibility of the RhRT photoreaction, these are the first time-resolved vibrational spectra of bathoRT 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 RhRT or bathoRT (potentially assignable to the blue-shifted intermediate, BSI). Excellent agreement is found between the major features of the RhRT and bathoRT vibrational spectra measured via PTR/CARS and earlier resonance Raman (RR) spectra taken at low temperatures (LT) selected to thermally stabilize (freeze) batho. Comparisons of the CC stretching mode region reveal a 12-cm-1 shift upon bathoRT 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 batho, thereby supporting an inverse relationship between CC frequencies and absorption maxima proposed for retinal proteins. Comparisons of the CC 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 batho, the direction of which is opposite to the blue shifts observed in the visible absorption maxima of Rh (7 nm) and batho (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 bathoRT structure than in the corresponding static bathoLT structure.
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