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 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.

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

The operation of bacteriorhodopsin (BR) from the archaeon Halobacterium salinarum is based on the photochromic reaction of isomerization of the chromophore group (the retinal protonated Schiff base, RPSB) from the all-trans to the 13-cis form. The ultrafast dynamics of the reverse 13-cis → all-trans photoreaction was studied using femtosecond transient absorption spectroscopy in comparison with the forward photoreaction. The forward photoreaction was initiated by photoexcitation of BR by pulse I (540 nm). The reverse photoreaction was initiated by photoexcitation of the product K₅₉₀ at an early stage of its formation (5 ps) by pulse II (660 nm). The conversion of the excited K₅₉₀ to the ground state proceeds at times of 0.19, 1.1, and 16 ps with the relative contributions of ~20/60/20, respectively. All these decay channels lead to the formation of the initial state of BR as a product with a quantum yield of ~1. This state is preceded by vibrationally excited intermediates, the relaxation of which occurs in the 16 ps time range. Likely, the heterogeneity of the excited state of K₅₉₀ is determined by the heterogeneity of its chromophore center. The forward photoreaction includes two components—0.52 and 3.5 ps, with the relative contributions of 91/9, respectively. The reverse photoreaction initiated from K₅₉₀ proceeds more efficiently in the conical intersection (CI) region but on the whole at a lower rate compared to the forward photoreaction, due to significant heterogeneity of the potential energy surface.

The review discusses experimental findings and theoretical studies of photoreversible transformations of retinal-containing proteins (rhodopsins). Understanding these processes is essential for elucidating the mechanisms of photochemical and conformational transformations of these proteins. Particular emphasis is placed on visual rhodopsins, as photoreversibility plays a crucial role in its physiological regeneration in the rhabdoms of invertebrates and the cone photoreceptors of the vertebrate retina. In recent years, interest in the mechanisms of photoreversible reactions in retinal-containing proteins has grown substantially, driven by their potential applications as active components in optoelectronic devices and biosensors.

Objective: Rhodopsin from the eubacterium Exiguobacterium sibiricum (ESR) performs the lightdependent proton pumping function. The operation of ESR is based on the ultrafast photochemical reaction of isomerization of the retinal chromophore, which triggers dark processes closed in a photocycle. Many parameters of the photocycle are determined by the presence of a hydrogen bond between the primary counterion Asp85 and the chromophore. ESR in detergent micelles pumps protons most efficiently at pH > 9, when such a bond is most probable. Methods: In the present study, the photochemical reaction of ESR at pH 9.5 was investigated by femtosecond laser absorption spectroscopy. Results and Discussion: It was shown that photoisomerization of the chromophore group occurs in 0.51 ps, with the contribution from the reactive excited state being ca. 80%. A comparison with the earlier obtained data for pH 7.4 showed that, at pH 9.5, the reaction proceeds much faster and more efficiently. Conclusions: The present results confirm the important role of the chromophore group counterion in the photoactivated processes of rhodopsins.