Vibrational Spectrum of Bathorhodopsin in the Room-Temperature Rhodopsin Photoreaction

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) batho. 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 batho, 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 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 batho RT structure than in the corresponding static batho LT structure.