The charge separation mechanism in spinach photosystem II core complexes (PSII-CC) and D1/D2/cytb559 reaction centers (D1D2-RC) was investigated using broadband femtosecond absorption spectroscopy in combination with kinetic modeling. The population dynamics of electronic states was analyzed based on decomposing the spectral-temporal matrices ΔA(λ,t) into contributions from excited states of chlorophyll (Chl) and difference spectra of the oxidized electron donor P680 and the reduced pheophytin acceptor PheoD1 obtained by the steady-state photoaccumulation technique. The results are discussed within the models, suggesting that in D1D2-RC at 279 K/77 K: (i) the predominant mechanism of primary charge separation is formation of the ChlD1+PheoD1- ion-radical pair in 3.6/7.6 ps followed by the hole transfer to P680 in 30/108 ps; (ii) the excitation energy transfer from ChlZ molecules to central RC pigments occurs within 19/28 ps, overlapping in time with the electron-transfer reactions; (iii) the free energy level of ChlD1+PheoD1- is higher than that of the relaxed exciton state by ∼30 meV, but the states become equally energetic during the ChlD1+ → P680 hole transfer. The charge separation kinetics in D1D2-RC at 77 K was found to be significantly heterogeneous. In PSII-CC at 279 K, (i) the apparent time of energy transfer from the excited antenna to RC is ∼60 ps, while the effective time of the ChlD1+PheoD1- formation in a small fraction of complexes with directly excited RC is comparable to that in D1D2-RC (3.7 ps); (ii) unlike D1D2-RC, the primary charge separation occurs with a decrease in free energy between RC⁎ and ChlD1+PheoD1-.

The function of rhodopsin of the psychrotrophic eubacterium Exiguobacterium sibiricum (ESR), found in permafrost soil, is light-dependent outward proton transport. This process is driven by photoisomerization of its chromophore group-the retinal protonated Schiff base (RPSB). The protonation state of the complex counterion of RPSB inside the protein environment significantly alters the efficiency of the ESR functioning. In this work, the pH dependence of photochemical reaction dynamics in ESR is studied using femtosecond transient absorption spectroscopy. Our results show that the photoreaction is most efficient at alkaline pH (9.5). Decrease in pH to acidic values (5.3) reduces the photoisomerization rate by 40%, and its efficiency drops by about 65%. This suppression arises from both the diminished productive decay from the reactive excited state and the enhanced population of a long-lived nonreactive excited state. Conformational sampling by metadynamics followed by quantum mechanics/molecular mechanics calculations provides evidence for structural heterogeneity of the ESR chromophore binding pocket. Multiple conformations can be distinguished by variations in hydrogen-bonding networks involving RPSB and its complex counterion (including residues D85, D221, H57, and water molecules). The change in the pH shifts the relative populations of the reactive and nonreactive conformations, thereby explaining the experimentally observed reduction in both photoisomerization rate and overall quantum yield under acidic conditions. Our findings highlight the important catalytic role played by the chromophore counterion in mediating the photoreaction dynamics of ESR and related microbial rhodopsins.

The sustainable functioning of the purified photosystem I (PSI) pigment-protein complex requires exogenous redox-mediators, facilitating the primary electron donor P 700 + reduction and terminal acceptor [F A /F B ] − oxidation. The redox couple ascorbate/2,6-dichlorophenolindophenol (Asc/DCPIP) was shown to be more efficient, than the couple Asc/ N,N,N',N' -tetramethyl- p -phenylenediamine (TMPD) both in photosynthetic studies and in biohybrid photovoltaic devices. We investigated the interactions of DCPIP with purified cyanobacterial PSI in the presence of Asc excess under laser flash excitation. Here we show that DCPIP, in contrast to TMPD, competes efficiently as an electron acceptor with the backward electron transfer from [F A /F B ] − to P 700 + even at micromolar concentrations, indicating accumulation of the oxidized DCPIP under aerobic conditions in the presence of Asc excess. The reduction of P 700 + includes contributions both from reduced DCPIP and semiquinone DCPIP •− . The rates of P 700 + reduction and [F A /F B ] − oxidation by DCPIP demonstrate complex pH-dependencies, related to changes in protonation state of the mediator and probably to redistribution of electron density between the terminal cofactors F A and F B . The rate constants of the electron transfer from Asc, DCPIP and TMPD to P 700 + and of the electron outflow from [F A /F B ] − to the oxidized forms of these compounds are estimated by kinetic modeling. The obtained data reveal thermodynamic, kinetic and electrostatic factors responsible for the high DCPIP efficiency as electron donor and acceptor for PSI.

The Orange Carotenoid Protein (OCP) is a unique water-soluble photoactive protein that plays a critical role in regulating the balance between light harvesting and photoprotective responses in cyanobacteria. The challenge in understanding OCP´s photoactivation mechanism stems from the heterogeneity of the initial configurations of its embedded ketocarotenoid, which in the dark-adapted state can form up to two hydrogen bonds to critical amino acids in the protein’s C-terminal domain, and the extremely low quantum yield of primary photoproduct formation. While a series of experiments involving point mutations within these contacts helped us to identify these challenges, they did not resolve them. To overcome this, we shifted from classical mutagenesis to the translational introduction of non-canonical amino acid residues into the OCP structure. In this work, we demonstrate that replacing a single meta-hydrogen in tyrosine-201 with a halogen atom (chlorine, bromine, or iodine) leads to targeted modifications in the keto-carotenoid-protein matrix interaction network, both in the dark-adapted state and upon photoactivation. We found that such atomic substitutions allow us to effectively weaken key hydrogen bonds without disrupting protein folding, thereby increasing the yield of OCP photoactivation products. Such genetically encoded chemical modification of individual atoms and their systematic in situ variation in complex protein structures establishes a foundation for transforming OCP into a practical tool for optogenetics and other applications.

Cyanobacteria use soluble antenna megacomplexes, phycobilisomes (PBSs), to maximize light-harvesting efficiency and small photoswitchable orange carotenoid proteins (OCPs) to down-regulate PBSs in high light. Among known PBS morphologies, the one from the basal cyanobacterial genus Gloeobacter still lacks detailed structural characterization. Here, we reconstructed a cryo-electron microscopy structure of the >10-megadalton Gloeobacter violaceus PBS, with diverging, conformationally mobile bundles of rods composed of stacked phycoerythrin and phycocyanin hexamers, stemming from a pentacylindrical allophycocyanin core belted by auxiliary phycocyanin hexamers. We show how two Gloeobacter-specific multidomain linker proteins, Glr1262 and Glr2806, maintain this bundle-shaped architecture and reveal its differential regulation via nonphotochemical quenching by two OCP types of G. violaceus that recognize separate binding sites within the allophycocyanin core, including lateral cylinders absent in tricylindrical cores.

Flavin-binding proteins (flavoproteins) are widespread in nature, revealing versatile oxidation-reduction reactions and photochemistry. Flavoproteins derived from LOV domains are used for engineering of ligh-tresponsive tools in optogenetics, as well as fluorescent markers and photogenerators of reactive oxygen species. Despite extensiev efforts, all currently used LOV-derived proteins have similar absorption spectra with maxima around 275, 35-0375, and 450-485 nm. Here, we describe the discovery of a large Stokes shift flavi-nbased fluorescent protein, LSSFbFP, which can be obtained in vivo and in vitro , with absorption maxima at 340-350 and 395-405 nm. Fluorescence emission of LSSFbFP mirrors that of classical FbFPs with the maximum at ~500 nm. We sho that the protein binds lumichrome as the chromophore and use low temperature and time-resolved spectroscopy, X-ray crystallography and modeling to prove that the apparent Stokes shift of LSSFbFP occurs due to excited state proton phenomena observed in flavoproteni s and pave the way for engineering of new flavin-based molecular instruments. ### Competing Interest Statement The authors have declared no competing interest. Ministry of Science and Higher Education of the Russian Federation, agreement 075-03-2025-662, project FSMG-2025-0003

In this study, the vibrational characteristics of optically excited echinenone in various solvents and the Orange Carotenoid Protein (OCP) in red and orange states are systematically investigated through steady-state and time-resolved spectroscopy techniques. Time-resolved experiments, employing both Transient Absorption (TA) and Femtosecond Stimulated Raman Spectroscopy (FSRS), reveal different states in the OCP photoactivation process. The time-resolved studies indicate vibrational signatures of exited states positioned above the S1 state during the initial 140 fs of carotenoid evolution in OCP, an absence of a vibrational signature for the relaxed S1 state of echinenone in OCP, and more robust signatures of a highly excited ground state (GS) in OCP. Differences in S1 state vibration population signatures between OCP and solvents are attributed to distinct conformations of echinenone in OCP and hydrogen bonds at the keto group forming a short-lived intramolecular charge transfer (ICT) state. The vibrational dynamics of the hot GS in OCP show a more pronounced red shift of ground state C=C vibration compared to echinenone in solvents, thus suggesting an unusually hot form of GS. The study proposes a hypothesis for the photoactivation mechanism of OCP, emphasizing the high level of excitation in longitudinal stretching modes as a driving force. In conclusion, the comparison of vibrational signatures reveals unique dynamics of energy dissipation in OCP, providing insights into the photoactivation mechanism and highlighting the impact of the protein environment on carotenoid behavior. The study underscores the importance of vibrational analysis in understanding the intricate processes involved in early phase OCP photoactivation.

Primary processes of light energy conversion by Photosystem II (PSII) were studied using femtosecond broadband pump-probe absorption difference spectroscopy. Transient absorption changes of core complexes isolated from the cyanobacterium Synechococcus sp. PCC 7335 grown under far-red light (FRL-PSII) were compared with the canonical Chl a containing spinach PSII core complexes upon excitation into the red edge of the Q(y) band. Absorption changes of FRL-PSII were monitored at 278 K in the 400-800 nm spectral range on a timescale of 0.1-500 ps upon selective excitation at 740 nm of four chlorophyll (Chl) f molecules in the light harvesting antenna, or of one Chl d molecule at the Chl(D1) position in the reaction center (RC) upon pumping at 710 nm. Numerical analysis of absorption changes and assessment of the energy levels of the presumed ion-radical states made it possible to identify P(D1)(+)Chl(D1)(-) as the predominant primary charge-separated radical pair, the formation of which upon selective excitation of Chl d has an apparent time of similar to 1.6 ps. Electron transfer to the secondary acceptor pheophytin Pheo(D1) has an apparent time of similar to 7 ps with a variety of excitation wavelengths. The energy redistribution between Chl a and Chl f in the antenna occurs within 1 ps, whereas the energy migration from Chl f to the RC occurs mostly with lifetimes of 60 and 400 ps. Potentiometric analysis suggests that in canonical PSII, P(D1)(+)Chl(D1)(-) can be partially formed from the excited (P(D1)Chl(D1))* state.

The exciton interaction of four chlorophyll a (Chl a) molecules in a symmetrical tetrameric complex of the watersoluble chlorophyll -binding protein BoWSCP was analyzed in the pH range of 3-11. Exciton splitting Delta E = 232 +/- 2 cm(-1) of the Qy band of Chl a into two subcomponents with relative intensities of 78.1 +/- 0.7 % and 21.9 +/- 0.7 % was determined by a joint decomposition of the absorption and circular dichroism spectra into Gaussian functions. The exciton coupling parameters were calculated based on the BoWSCP atomic structure in three approximations: the point dipole model, the distributed atomic monopoles, and direct ab initio calculations in the TDDFT/PCM approximation. The Coulomb interactions of monomers were calculated within the continuum model using three values of optical permittivity. The models based on the properties of free Chl a in solution suffer from significant errors both in estimating the absolute value of the exciton interaction and in the relative intensity of exciton transitions. Calculations within the TDDFT/PCM approximation reproduce the experimentally determined parameters of the exciton splitting and the relative intensities of the exciton bands. The following factors of pigment-protein and pigment-pigment interactions were examined: deviation of the macrocycle geometry from the planar conformation of free Chl; the formation of hydrogen bonds between the macrocycle and water molecules; the overlap of wave functions of monomers at close distances. The most significant factor is the geometrical deformation of the porphyrin macrocycle, which leads to an increase in the dipole moment of Chl monomer from 5.5 to 6.9 D and to a rotation of the dipole moment by 15 degrees towards the cyclopentane ring. The contributions of resonant charge-transfer states to the wave functions of the Chl dimer were determined and the transition dipole moments of the symmetric and antisymmetric charge-transfer states were estimated.

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.

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

The conversion of light energy in the near-infrared spectral range by photosynthetic pigment–protein complexes has been intensively studied in recent years in connection with the discovery of cyanobacteria, in the photosynthetic apparatus of which chlorophylls f and d absorbing long-wavelength light are present. Acaryochloris marina occupies a special place among such cyanobacteria. Its photosystem I contains mainly chlorophyll d; it is a part of a special P740 pair, the absorption spectrum of which is shifted to the red region by 40 nm. This causes a decrease in the energy of the excited state of P740 by 0.1 eV compared with photosystem I containing chlorophyll a. Complexes of photosystem I from A. marina also have the following peculiarities: four chlorophyll a molecules of the reaction center are replaced by chlorophyll d, and the third pair of chlorophyll a molecules involved in electron transfer are replaced by pheophytin a. The presence of spectrally different cofactors (chlorophyll d and pheophytin a) made it possible to reliably establish the intermediates of primary charge separation reactions in this photosystem I. In this review, we considered the latest results of studies of photochemical energy conversion in the reaction centers of photosystem I of A. marina and possible mechanisms for compensating energy losses of the use of low-energy far-red light for photosynthesis.

Primary excitation energy transfer and charge separation in photosystem I (PSI) from the extremophile desert green alga Chlorella ohadii grown in low light were studied using broadband femtosecond pump-probe spectroscopy in the spectral range from 400 to 850 nm and in the time range from 50 fs to 500 ps. Photochemical reactions were induced by the excitation into the blue and red edges of the chlorophyll Qy absorption band and compared with similar processes in PSI from the cyanobacterium Synechocystis sp. PCC 6803. When PSI from C. ohadii was excited at 660 nm, the processes of energy redistribution in the light-harvesting antenna complex were observed within a time interval of up to 25 ps, while formation of the stable radical ion pair P700+A1− was kinetically heterogeneous with characteristic times of 25 and 120 ps. When PSI was excited into the red edge of the Qy band at 715 nm, primary charge separation reactions occurred within the time range of 7 ps in half of the complexes. In the remaining complexes, formation of the radical ion pair P700+A1− was limited by the energy transfer and occurred with a characteristic time of 70 ps. Similar photochemical reactions in PSI from Synechocystis 6803 were significantly faster: upon excitation at 680 nm, formation of the primary radical ion pairs occurred with a time of 3 ps in 30

Phycobilisomes (PBSs) are giant water-soluble light-harvesting complexes of cyanobacteria and red algae, consisting of hundreds of phycobiliproteins precisely organized to deliver the energy of absorbed light to chlorophyll chromophores of the photosynthetic electron-transport chain. Quenching the excess of excitation energy is necessary for the photoprotection of photosynthetic apparatus. In cyanobacteria, quenching of PBS excitation is provided by the Orange Carotenoid Protein (OCP), which is activated under high light conditions. In this work, we describe parameters of anti-Stokes fluorescence of cyanobacterial PBSs in quenched and unquenched states. We compare the fluorescence readout from entire phycobilisomes and their fragments. The obtained results revealed the heterogeneity of conformations of chromophores in isolated phycobiliproteins, while such heterogeneity was not observed in the entire PBS. Under excitation by low-energy quanta, we did not detect a significant uphill energy transfer from the core to the peripheral rods of PBS, while the one from the terminal emitters to the bulk allophycocyanin chromophores is highly probable. We show that this direction of energy migration does not eliminate fluorescence quenching in the complex with OCP. Thus, long-wave excitation provides new insights into the pathways of energy conversion in the phycobilisome.

This paper focuses on the energetics of photoinduced charge separation reactions between closely spaced molecules of chlorophyll (Chl) and pheophytin (Pheo). The reaction centers of photosystems PSI and PSII include three pairs of spectrally similar porphyrin cofactors, whose structure allows the implementation of alternative mechanisms of primary charge separation. A continuum model for the formation of ion-radical pairs in a dielectric environment based on partial charges calculated ab initio is considered. The model describes the experimental variation of the midpoint redox potentials of Chl and Pheo in solutions with different permittivities. Within this model, the formation energy of primary ion-radical pairs is estimated for the alternative mechanisms of charge separation in PSI and PSII discussed in the literature. In the considered approximation in PSII, the primary charge separation between the monomer of Chl (Chl D1 ) and Pheo (Pheo D1 ) is the only energetically allowed mechanism. The absence of Pheo in the same position in the reaction center of PSI denies the possibility of the Chl monomer in this complex acting as the primary electron donor. Stabilization of the primary ion-radical pair in PSI can occur due to the delocalization of the electron density along the dimer of a special pair of Chl molecules (Р 700 ) and the heterodimer of Chl molecules forming the primary acceptor A 0 .

The paper reports on the absorption dynamics of chlorophyll a in a symmetric tetrameric complex of the water-soluble chlorophyll-binding protein BoWSCP. It was measured by a broadband femtosecond laser pump-probe spectroscopy within the range from 400 to 750 nm and with a time resolution of 20 fs-200 ps. When BoWSCP was excited in the region of the Soret band at a wavelength of 430 nm, nonradiative intramolecular conversion S 3 →S 1 was observed with a characteristic time of 83 ± 9 fs. When the complex was excited in the region of the Q y band at 670 nm, relaxation transition between two excitonic states of the chlorophyll dimer was observed in the range of 105 ± 10 fs. Absorption spectra of the excited singlet states S 1 and S 3 of chlorophyll a were obtained. The delocalization of the excited state between exciton-coupled Chl molecules in BoWSCP tetramer changed in time and depended on the excitation energy. When BoWSCP is excited in the Soret band region, an ultrafast photochemical reaction is observed. This could result from the reduction of tryptophan in the vicinity of chlorophyll.

The absorption dynamics of chlorophyll a in a symmetric tetrameric complex of the water-soluble chlorophyll-binding protein BoWSCP was measured by the broadband femtosecond laser pump-probe spectroscopy within the range from 400 to 780 nm with a time resolution of 20 f-200 ps. When BoWSCP was excited in the region of the Soret band at a wavelength of 430 nm, a nonradiative intramolecular conversion S3 → S1 was observed with a characteristic time of 83 ± 9 fs. When the complex was excited in the region of the Qy band at a wavelength of 670 nm, a relaxation transition between two excitonic states of the chlorophyll dimer was observed with a time of 105 ± 10 fs. Absorption spectra of excited singlet states S1 and S3 of chlorophyll a were obtained. It has been demonstrated that the delocalization of the excited state between exciton-coupled Chl molecules in the BoWSCP tetramer changes in time and depends on the excitation energy. Upon excitation of BoWSCP, an ultrafast photochemical reaction is observed in the Soret region, apparently due to the reduction of tryptophan in the vicinity of chlorophyll.

The dependence of the dipole moment of chlorophyll a ’s (Chl) S 0 → S 1 transition on the value of the solvent refractive index n is calculated. The interactions between the electric field of a light wave, the electronic transition of the pigment to an excited state, and the dielectric polarization of an optical medium are analyzed. The reactive changes in Chl’s transition dipole moment in solvents with different refractive index values are calculated in the time-dependent density functional theory (TD–DFT) using the LC-ωPBE hybrid functional and the polarizable continuum model. The ab initio calculations are approximated by the Onsager reactive field model with an effective polarizability of Chl equal to 21 Å 3 . The model quantitatively describes the experimental dependence of Chl’s extinction coefficient in solvents with a refractive index of 1.3 < n < 1.7. In a protein environment with a refractive index of n = 1.4, the transition dipole moment of Chl is 5.5 D. For this environment, the distributions of the electrostatic potential in the ground and excited states of Chl are calculated; the ab initio calculations are approximated by a set of partial transient charges located on the heavy atoms of the π-conjugated system of the Chl molecule.

Проведен расчет зависимости дипольного момента перехода S 0 → S 1 хлорофилла a от величины индекса рефракции n растворителя. Проанализированы взаимодействия между электрическим полем световой волны, электронным переходом пигмента в возбужденное состояние и диэлектрической поляризацией оптической среды. Эффект реактивного увеличения переходного дипольного момента молекулы хлорофилла a в растворителях с различной величиной индекса рефракции рассчитан в рамках нестационарной теории функционала плотности (TD–DFT) с использованием гибридного функционала LC-ωPBE и модели поляризуемого континуума. Расчеты ab initio аппроксимированы моделью реактивного поля Онзагера с эффективной поляризуемостью хлорофилла равной 21 Å 3 . Модель количественно описывает экспериментальную зависимость коэффициента экстинкции хлорофилла a в растворителях с индексом рефракции 1.3 < n < 1.7. В белковом окружении с индексом рефракции n = 1.4 величина дипольного момента перехода хлорофилла составляет 5.5 Д. Для этого окружения было рассчитано распределение электростатического потенциала в основном и возбужденном состояниях хлорофилла; расчеты ab initio аппроксимированы набором парциальных переходных зарядов, расположенных на тяжелых атомах π-сопряженной системы молекулы хлорофилла.