The coherent 11-cis-retinal photoisomerization dynamics in bovine rhodopsin was studied by femtosecond time-resolved laser absorption spectroscopy at 30-fs resolution. Femtosecond pulses of 500, 535, and 560 nm wavelength were used for rhodopsin excitation to produce different initial Franck-Condon states and relevant distinct values of the vibrational energy of the molecule in its electron excited state. Time evolution of the photoinduced rhodopsin absorption spectra was monitored after femtosecond excitation in the spectral range of 400-720 nm. Oscillations of the time-resolved absorption signals of rhodopsin photoproducts represented by photorhodopsin(570) with vibrationally-excited all-trans-retinal and rhodopsin(498) in its initial state with vibrationally-excited 11-cis-retinal were studied. These oscillations reflect the dynamics of coherent vibrational wave-packets in the ground state of photoproducts. Fourier analysis of these oscillatory components has revealed frequencies, amplitudes, and initial phases of different vibrational modes, along which the motion of wave-packets of both photoproducts occurs. The main vibrational modes established are 62, 160 cm(-1) and 44, 142 cm(-1) for photorhodopsin(570) and for rhodopsin(498), respectively. These vibrational modes are directly involved in the coherent reaction under the study, and their amplitudes in the power spectrum obtained through the Fourier transform of the kinetic curves depend on the excitation wavelength of rhodopsin.

Ultrafast reverse photoreaction of visual pigment rhodopsin in the femtosecond time range at room temperature is demonstrated. Femtosecond two-pump probe experiments with a time resolution of 25 fs have been performed. The first рump pulse at 500 nm initiated cis-trans photoisomerization of rhodopsin chromophore, 11-cis retinal, which resulted in the formation of the primary ground-state photoproduct within a mere 200 fs. The second pump OPEN ACCESSMolecules 2014, 19 18352 pulse at 620 nm with a varying delay of 200 to 3750 fs relative to the first рump pulse, initiated the reverse phototransition of the primary photoproduct to rhodopsin. The results of this photoconversion have been observed on the differential spectra obtained after the action of two pump pulses at a time delay of 100 ps. It was found that optical density decreased at 560 nm in the spectral region of bathorhodopsin absorption and increased at 480 nm, where rhodopsin absorbs. Rhodopsin photoswitching efficiency shows oscillations as a function of the time delay between two рump pulses. The quantum yield of reverse photoreaction initiated by the second pump pulse falls within the range 15% ± 1%. The molecular mechanism of the ultrafast reversible photoreaction of visual pigment rhodopsin may be used as a concept for the development of an ultrafast optical molecular switch.

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The primary stages of the Exiguobacterium sibiricum rhodopsin (ESR) photocycle were investigated by femtosecond absorption laser spectroscopy in the spectral range of 400−900 nm with a time resolution of 25 fs. The dynamics of the ESR photoreaction were compared with the reactions of bacteriorhodopsin (bR) in purple membranes (bR PM ) and in recombinant form (bR rec ). The primary intermediates of the ESR photocycle were similar to intermediates I, J, and K in bacteriorhodopsin photoconversion. The CONTIN program was applied to analyze the characteristic times of the observed processes and to clarify the reaction scheme. A similar photoreaction pattern was observed for all studied retinal proteins, including two consecutive dynamic Stokes shift phases lasting ∼0.05 and ∼0.15 ps. The excited state decays through a femtosecond reactive pathway, leading to retinal isomerization and formation of product J, and a picosecond nonreactive pathway that leads only to the initial state. Retinal photoisomerization in ESR takes 0.69 ps, compared with 0.48 ps in bR PM and 0.74 ps in bR rec . The nonreactive excited state decay takes 5 ps in ESR and ∼3 ps in bR. We discuss the similarity of the primary reactions of ESR and other retinal proteins.

Photochemical reaction dynamics of the primary events in recombinant bacteriorhodopsin (bR) was studied by femtosecond laser absorption spectroscopy with 25-fs time resolution. bR was produced in an Escherichia coli expression system. Since bR was prepared in a DMPC-CHAPS micelle system in the monomeric form, its comparison with trimeric and monomeric forms of the native bacteriorhodopsin (bR and bR, respectively) was carried out. We found that bR intermediate I (excited state of bR) was formed in the range of 100 fs, as in the case of bR and bR. Further processes, namely the decay of the excited state I and the formation of intermediates J and K of bR, occurred more slowly compared to bR, but similarly to bR. The lifetime of intermediate I, judging from the signal of ΔA(470-480 nm), was 0.68 ps (78%) and 4.4 ps (22%) for bR, 0.52 ps (73%) and 1.7 ps (27%) for bR, and 0.45 ps (90%) and 1.75 ps (10%) for bR. The formation time of intermediate K, judging from the signal of ΔA(625-635 nm), was 13.5 ps for bR, 9.8 ps for bR, and 4.3 ps for bR. In addition, there was a decrease in the photoreaction efficiency of bR and bR as seen by a decrease in absorbance in the differential spectrum of the intermediate K by ~14%. Since photochemical properties of bR are similar to those of the monomeric form of the native protein, bR and its mutants can be considered as a basis for further studies of the mechanism of bacteriorhodopsin functioning.