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The kinetics of the primary electron donor P 700 + and the quinone acceptor A 1 - redox transitions were simultaneously studied for the first time in the time range of 200 μs-10 ms using high-frequency pulse Q-band EPR spectroscopy at cryogenic temperatures in various complexes of photosystem I (PSI) from the cyanobacterium Synechocystis sp. PCC 6803. In the A 1 -core PSI complexes that lack 4Fe4S clusters, the kinetics of the A 1 - and P 700 + signals disappearance at 100 K were similar and had a characteristic time of τ ≈ 500 μs, caused by charge recombination in the P 700 + A 1A - ion-radical pair in the A branch of redox cofactors. The kinetics of the backward electron transfer from A 1B - to P 700 + in the B branch of redox cofactors with τ < 100 μs could not be resolved due to time limitations of the method. In the native PSI complexes with a full set of redox cofactors and in the F X -core complexes, containing the 4Fe4S cluster F X , the kinetics of the A 1 - signal was significantly faster than that of the P 700 + signal. The disappearance of the A 1 - signal had a characteristic time of 280-350 μs; it was suggested that, in addition to the backward electron transfer from A 1A - to P 700 + with τ ≈ 500 μs, its kinetics also includes the forward electron transfer from A 1A - to the 4Fe4S cluster F X , which had slowed down to 150-200 μs. In the kinetics of P 700 + reduction, it was possible to distinguish components caused by the backward electron transfer from A 1 -  (τ ≈ 500 μs) and from 4Fe4S clusters (τ = 1 ms for the F X -core and τ > 5 ms for native complexes). These results are in qualitative agreement with the data on the kinetics of P 700 + reduction obtained previously using pulse absorption spectrometry at cryogenic temperatures.

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 ∼1.6 ps. Electron transfer to the secondary acceptor pheophytin Pheo D1 has an apparent time of ∼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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

The widespread use of disinfectants and antiseptics, and consequently their release into the environment, determines the relevance of studying their potential impact on the main producers of organic matter on the planet-photosynthetic organisms. The review examines the effects of some biguanides and quaternary ammonium compounds, octenidine, miramistin, chlorhexidine, and picloxidine, on the functioning of the photosynthetic apparatus of various organisms (Strakhovskaya et al. in Photosynth Res 147:197-209, 2021; Knox et al. in Photosynth Res 153:103, 2022; Paschenko et al. in Photosynth Res 155:93-105, 2023a, Photosynth Res 2023b). A common feature of these antiseptics is the combination of hydrophobic and hydrophilic regions in the molecules, the latter carrying a positive charge(s). The comparison of the results obtained with intact bacterial membrane vesicles (chromatophores) and purified pigment-protein complexes (photosystem II and I) of oxygenic organisms allows us to draw conclusions about the mechanisms of the cationic antiseptic action on the functional properties of the components of the photosynthetic apparatus., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

In this study, the effects of cationic antiseptics such as chlorhexidine, picloxidine, miramistin, and octenidine at concentrations up to 150 µM on fluorescence spectra and its lifetimes, as well as on light-induced electron transfer in protein-pigment complexes of photosystem I (PSI) isolated from cyanobacterium Synechocystis sp. PCC 6803 have been studied. In doing so, octenidine turned out to be the most "effective" in terms of its influence on the spectral and functional characteristics of PSI complexes. It has been shown that the rate of energy migration from short-wavelength forms of light-harvesting chlorophyll to long-wavelength ones slows down upon addition of octenidine to the PSI suspension. After photo-separation of charges between the primary electron donor P 700 and the terminal iron-sulfur center(s) F A /F B , the rate of forward electron transfer from (F A /F B ) - to the external medium slows down while the rate of charge recombination between reduced F A /F B - and photooxidized P 700 + increases. The paper considers the possible causes of the observed action of the antiseptic., (© 2023. The Author(s), under exclusive licence to Springer Nature B.V.)

Measurement of electrical potential difference (Δψ) in membrane vesicles (chromatophores) from the purple bacterium Rhodobacter sphaeroides associated with the surface of a nitrocellulose membrane filter (MF) impregnated with a phospholipid solution in decane or immersed into it in the presence of exogenous mediators and disaccharide trehalose demonstrated an increase in the amplitude and stabilization of the signal under continuous illumination. The mediators were the ascorbate/N,N,N'N'-tetramethyl-p-phenylenediamine pair and ubiquinone-0 (electron donor and acceptor, respectively). Although stabilization of photoelectric responses upon long-term continuous illumination was observed for both variants of chromatophore immobilization, only the samples immersed into the MF retained the functional activity of reaction centers (RCs) for a month when stored in the dark at room temperature, which might be due to the preservation of integrity of chromatophore proteins inside the MF pores. The stabilizing effect of the bioprotector trehalose could be related to its effect on both the RC proteins and the phospholipid bilayer membrane. The results obtained will expand current ideas on the use of semi-synthetic structures based on various intact photosynthetic systems capable of converting solar energy into its electrochemical form.

Retinal-containing light-sensitive proteins - rhodopsins - are found in many microorganisms. Interest in them is largely explained by their role in light energy storage and photoregulation in microorganisms, as well as the prospects for their use in optogenetics to control neuronal activity, including treatment of various diseases. One of the representatives of microbial rhodopsins is ESR, the retinal protein of Exiguobacterium sibiricum. What distinguishes ESR from homologous proteins is the presence of a lysine residue (Lys96) as a proton donor for the Schiff base. This feature, along with the hydrogen bond of the proton acceptor Asp85 with the His57 residue, determines functional characteristics of ESR as a proton pump. This review examines the results of ESR studies conducted using various methods, including direct electrometry. Comparison of the obtained data with the results of structural studies and with other retinal proteins allows us to draw conclusions about the mechanisms of transport of hydrogen ions in ESR and similar retinal proteins.

Chromatophores (Chr) from photosynthetic nonsulfur purple bacterium Rhodobacter sphaeroides immobilized onto a Millipore membrane filter (MF) and sandwiched between two semiconductor indium tin oxide (ITO) electrodes (termed ITO|Chr - MF|ITO) have been used to measure voltage (ΔV) induced by continuous illumination. The maximum ΔV was detected in the presence of ascorbate / N,N,N'N'-tetramethyl-p-phenylenediamine couple, coenzyme UQ 0 , disaccaride trehalose and antimycin A, an inhibitor of cytochrome bc 1 complex. In doing so, the light-induced electron transfer in the reaction centers was the major source of photovoltages. The stability of the voltage signal upon prolonged irradiation (>1 h) may be due to the maintenance of a conformation that is optimal for the functioning of integral protein complexes and stabilization of lipid bilayer membranes in the presence of trehalose. Retaining ∼70 % of the original photovoltage performance on the 30th day of storage at 23 °C in the dark under air was achieved after re-injection of fresh buffer (∼40 μL) containing redox mediators into the ITO|Chr - MF|ITO system. The approach we use is easy and can be extended to other biological intact systems (cells, thylakoid membranes) capable of converting energy of light., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier B.V.)

Microbial rhodopsins comprise a diverse family of retinal-containing membrane proteins that convert absorbed light energy to transmembrane ion transport or sensory signals. Incorporation of these proteins in proteoliposomes allows their properties to be studied in a native-like environment; however, unidirectional protein orientation in the artificial membranes is rarely observed. We aimed to obtain proteoliposomes with unidirectional orientation using a proton-pumping retinal protein from Exiguobacterium sibiricum , ESR, as a model. Three ESR hybrids with soluble protein domains (mCherry or thioredoxin at the C-terminus and Caf1M chaperone at the N-terminus) were obtained and characterized. The photocycle of the hybrid proteins incorporated in proteoliposomes demonstrated a higher pK a of the M state accumulation compared to that of the wild-type ESR. Large negative electrogenic phases and an increase in the relative amplitude of kinetic components in the microsecond time range in the kinetics of membrane potential generation of ESR-Cherry and ESR-Trx indicate a decrease in the efficiency of transmembrane proton transport. On the contrary, Caf-ESR demonstrates a native-like kinetics of membrane potential generation and the corresponding electrogenic stages. Our experiments show that the hybrid with Caf1M promotes the unidirectional orientation of ESR in proteoliposomes.

Herein, the effect of cationic antiseptics (chlorhexidine, picloxidine, miramistin, octenidine) on the initial processes of the delivery of light energy and its efficient use by the reaction centers in intact spinach photosystem II core complexes has been investigated. The characteristic effects-an increase in the fluorescence yield of light-harvesting pigments and a slowdown in the rate of energy migration in bacterial photosynthetic chromatophores has been recently demonstrated mainly in the presence of octenidine (Strakhovskaya et al., in Photosynth Res 147:197-209, 2021; Knox et al., in Photosynth Res, https://doi.org/10.1007/s11120-022-00909-8 , 2022). In this study, we also observed that in the presence of octenidine, the fluorescence intensity of photosystem II core complexes increases by 5-10 times, and the rate of energy migration from antennae to the reaction centers decreases by 3 times. In addition, with an increase in the concentration of this antiseptic, a new effect related to a shift of the spectrum, absorption and fluorescence to the short-wavelength region has been found. Similar effects were observed when detergent Triton X-100 was added to photosystem II samples. We concluded that the antiseptic primarily affects the structure of the internal light-harvesting antenna (CP43 and CP47), through which the excitation energy is delivered to the reaction center. As a result of such an impact, the chlorophyll molecules in this structure are destabilized and their optical and functional characteristics change., (© 2022. The Author(s), under exclusive licence to Springer Nature B.V.)

In photosynthetic reaction centers of intact photosystem I (PSI) complexes from cyanobacteria, electron transfer at room temperature occurs along two symmetrical branches of redox cofactors A and B at a ratio of ~3 : 1 in favor of branch A. Previously, this has been indirectly demonstrated using pulsed absorption spectroscopy and more directly by measuring the decay modulation frequencies of electron spin echo signals (electron spin echo envelope modulation, ESEEM), which allows to determine the distance between the separated charges of the primary electron donor P 700 + and phylloquinone acceptors A 1A - and A 1B - in the symmetric redox cofactors branches A and B. In the present work, these distances were determined using ESEEM in PSI complexes lacking three 4Fe-4S clusters, F X , F A , and F B , and the PsaC protein subunit (the so-called P 700 -A 1 core), in which phylloquinone molecules A 1A and A 1B serve as the terminal electron acceptors. It was shown that in the P 700 -A 1 core preparations, the average distance between the centers of the P 700 + A 1 - ion-radical pair at a temperature of 150 K in an aqueous glycerol solution and in a dried trehalose matrix, as well as in a trehalose matrix at 280 K, is ~25.5 Å, which corresponds to the symmetrical electron transfer along the A and B branches of redox cofactors at a ratio of 1 : 1. Possible reasons for the change in the electron transfer symmetry in PSI upon removal of the PsaC subunit and 4Fe-4S clusters F X , F A , and F B are discussed.

Transient absorption dynamics of chlorophylls a and d dissolved in tetrahydrofuran was measured by the broadband femtosecond laser pump-probe spectroscopy in a spectral range from 400 to 870 nm. The absorption spectra of the excited S 1 singlet states of chlorophylls a and d were recorded, and the dynamics of the of the Q y band shift of the stimulated emission (Stokes shift of fluorescence) was determined in a time range from 60 fs to 4 ps. The kinetics of the intramolecular conversion Q x →Q y (electronic transition S 2 →S 1 ) was measured; the characteristic relaxation time was 54 ± 3 and 45 ± 9 fs for chlorophylls a and d, respectively.

Light-driven proton transport by microbial retinal proteins such as archaeal bacteriorhodopsin involves carboxylic residues as internal proton donors to the catalytic center which is a retinal Schiff base (SB). The proton donor, Asp96 in bacteriorhodopsin, supplies a proton to the transiently deprotonated Schiff base during the photochemical cycle. Subsequent proton uptake resets the protonated state of the donor. This two step process became a distinctive signature of retinal based proton pumps. Similar steps are observed also in many natural variants of bacterial proteorhodopsins and xanthorhodopsins where glutamic acid residues serve as a proton donor. Recently, however, an exception to this rule was found. A retinal protein from Exiguobacterium sibiricum, ESR, contains a Lys residue in place of Asp or Glu, which facilitates proton transfer from the bulk to the SB. Lys96 can be functionally replaced with the more common donor residues, Asp or Glu. Proton transfer to the SB in the mutants containing these replacements (K96E and K96D/A47T) is much faster than in the proteins lacking the proton donor (K96A and similar mutants), and in the case of K96D/A47T, comparable with that in the wild type, indicating that carboxylic residues can replace Lys96 as proton donors in ESR. We show here that there are important differences in the functioning of these residues in ESR from the way Asp96 functions in bacteriorhodopsin. Reprotonation of the SB and proton uptake from the bulk occur almost simultaneously during the M to N transition (as in the wild type ESR at neutral pH), whereas in bacteriorhodopsin these two steps are well separated in time and occur during the M to N and N to O transitions, respectively., Competing Interests: Declaration of Competing Interest The authors declare no conflict of interest., (Copyright © 2022 Elsevier B.V. All rights reserved.)

This review analyzes new data on the mechanism of ultrafast reactions of primary charge separation in photosystem I (PS I) of cyanobacteria obtained in the last decade by methods of femtosecond absorption spectroscopy. Cyanobacterial PS I from many species harbours 96 chlorophyll a (Chl a ) molecules, including six specialized Chls denoted Chl 1A /Chl 1B (dimer P 700 , or P A P B ), Chl 2A /Chl 2B , and Chl 3A /Chl 3B arranged in two branches, which participate in electron transfer reactions. The current data indicate that the primary charge separation occurs in a symmetric exciplex, where the special pair P 700 is electronically coupled to the symmetrically located monomers Chl 2A and Chl 2B , which can be considered together as a symmetric exciplex Chl 2A P A P B Chl 2B with the mixed excited (Chl 2A P A P B Chl 2B ) * and two charge-transfer states P 700 + Chl 2A - and P 700 + Chl 2B - . The redistribution of electrons between the branches in favor of the A -branch occurs after reduction of the Chl 2A and Chl 2B monomers. The formation of charge-transfer states and the symmetry breaking mechanisms were clarified by measuring the electrochromic Stark shift of β-carotene and the absorption dynamics of PS I complexes with the genetically altered Chl 2B or Chl 2A monomers. The review gives a brief description of the main methods for analyzing data obtained using femtosecond absorption spectroscopy. The energy levels of excited and charge-transfer intermediates arising in the cyanobacterial PS I are critically analyzed., Competing Interests: Conflict of interestThe authors have no relevant financial or non-financial interests to disclose., (© International Union for Pure and Applied Biophysics (IUPAB) and Springer-Verlag GmbH Germany, part of Springer Nature 2022, Springer Nature or its licensor holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.)

Microbial rhodopsins are the family of retinal-containing proteins that perform primarily the light-driven transmembrane ion transport and sensory functions. They are widely distributed in nature and can be used for optogenetic control of the cellular activities by light. Functioning of microbial rhodopsins results in generation of the transmembrane electric potential in response to a flash that can be measured by direct time-resolved electrometry. This method was developed by L. Drachev and his colleagues at the Belozersky Institute and successfully applied in the functional studies of microbial rhodopsins. First measurements were performed using bacteriorhodopsin from Halobacterium salinarum -the prototype member of the microbial retinal protein family. Later, direct electrometric studies were conducted with proteorhodopsin from Exiguobacterium sibiricum (ESR), the sodium pump from Dokdonia , and other proteins. They allowed detailed characterization of the charge transfer steps during the photocycle of microbial rhodopsins and provided new insights for profound understanding of their mechanism of action., Competing Interests: Competing interestsThe authors declare no competing interests., (© International Union for Pure and Applied Biophysics (IUPAB) and Springer-Verlag GmbH Germany, part of Springer Nature 2022, Springer Nature or its licensor holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.)

This review provides a brief description of the structure and transport function of the recently discovered family of retinal-containing Na+-translocating rhodopsins. The main emphasis is put on the kinetics of generation of electric potential difference in the membrane during a single transporter turnover. According to the proposed transport mechanism of Na+-rhodopsin, the driving force for the Na+ translocation from the cytoplasm is the local electric field created by the H+ movement from the Schiff base.

In this minireview, we consider the methods of measurements of the light-induced steady state transmembrane electric potential (Δψ) generation by photosynthetic systems, e.g. photosystem I (PS I). The microelectrode technique and the detection of electrochromic bandshifts of carotenoid pigments are most appropriate for Δψ measurements in situ and in vivo. Direct electrometrical method and Δψ measurements in the photovoltaic system based on membrane filter (MF) sandwiched between semiconductor indium tin oxide electrodes (ITO) are suitable for studies of isolated pigment-protein complexes and small natural vesicles-chromatophores. In the presence of trehalose, ITO|PS I-MF|ITO system allows to keep a steady state level of ∆ψ after 1 h of illumination. According to preliminary experiments, this system is capable of providing steady state light-induced ∆ψ after several months of storage in the dark at room temperature under controlled humidity in the presence of trehalose. The long-term generation of light-induced ∆ψ in relatively simple system may serve as a source of the solar-to-electric energy conversion., Competing Interests: Competing interestsThe authors declare no competing interests., (© International Union for Pure and Applied Biophysics (IUPAB) and Springer-Verlag GmbH Germany, part of Springer Nature 2022.)

In this work, we investigated the redox transients of a number of water-soluble spin labels upon their interactions with Photosystem II (PS II) core complexes isolated from spinach leaves. We have found that the reactivity of nitroxide radicals, determined by the rate of their reduction upon illumination of PS II, depends on the chemical structure of radicals and the capability of their coming close to low-potential redox centers of photoactive PS II complexes. An enhanced capability of nitroxide radicals to accept electrons from PS II correlates with their chemical structure. Nitroxide radicals NTI (2,2,5,5-tetramethyl-4-nitromethylene-3-imidazolidine- N -oxyl) and Tacet (4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl-acetate), containing polar groups, appear to be most efficient acceptors of electrons donated by PS II compared to neutral (TEMPOL, 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl) or positively charged (Tamine, 4-amino-2,2,6,6-tetramethylpiperidine-l-oxyl) spin labels. We assume that enhanced reactivities of polar nitroxide radicals, NTI and Tacet, are determined (1) by their relatively high redox potentials, providing the possibility to accept electrons from PS II, and (2) by their affinities to the closest binding sites on the surface of PS II in the vicinity of the primary plastoquinone acceptor PQ A (12-14 Å) or/and in the intraprotein cavity for the secondary plastoquinone PQ B (~ 22 Å)., Competing Interests: Conflict of InterestThe authors declare that they have no conflict of interest., (© The Author(s), under exclusive licence to Springer-Verlag GmbH Austria, part of Springer Nature 2021.)

The effect of exogenous cytochrome c (cyt c) on kinetics of photoelectric responses (Δψ) of two types of photosystem II (PSII) core complexes (intact - PSII with active water-oxidizing complex and Mn-depleted complex) reconstituted into liposomes has been investigated by direct electrometric technique. PSII complexes were localized in the proteoliposome membranes with their donor side outward. An additional electrogenic phase was observed in the kinetics of Δψ generation in response to a laser flash besides the main fast (<0.3 µs) electrogenic component due to electron transfer from the redox-active tyrosine Y Z to the primary quinone acceptor Q A in the presence of oxidized cyt c (cyt c 3+ ) entrapped in the internal space of proteoliposomes with intact PSII complexes. This component with characteristic time τ ≈ 40 µs and relative amplitude of ~10% of the total Δψ was attributed to the vectorial electron transfer from Q A - to cyt c 3+ serving as an external acceptor. An additional electrogenic component with τ ~ 70 µs and a relative amplitude of ~20% of the total Δψ also appeared in the kinetics of Δψ formation, when cyt c 2+ was added to the suspension of proteoliposomes containing Mn-depleted PSII core complexes. This component was attributed to the electrogenic transfer of an electron from cyt c 2+ to photooxidized tyrosine Y Z . These data imply that cyt c 3+ serves as a very effective exogenous electron acceptor for Q A - in the case of intact PSII core complexes, and cyt c 2+ is an extremely efficient artificial electron donor for Y Z in the Mn-depleted PSII. The obtained data on the roles of cyt c 2+ and cyt c 3+ as an electron donor and acceptor for PSII, respectively, can be used to develop hybrid photoelectrochemical solar energy-converting systems based on photosynthetic pigment-protein complexes.

The kinetics of flash-induced re-reduction of the Photosystem II (PS II) primary electron donor P 680 was studied in solution and in trehalose glassy matrices at different relative humidity. In solution, and in the re-dissolved glass, kinetics were dominated by two fast components with lifetimes in the range of 2-7 μs, which accounted for >85% of the decay. These components were ascribed to the direct electron transfer from the redox-active tyrosine Y Z to P 680 + . The minor slower components were due to charge recombination between the primary plastoquinone acceptor Q A - and P 680 + . Incorporation of the PS II complex into the trehalose glassy matrix and its successive dehydration caused a progressive increase in the lifetime of all kinetic phases, accompanied by an increase of the amplitudes of the slower phases at the expense of the faster phases. At 63% relative humidity the fast components contribution dropped to ~50%. A further dehydration of the trehalose glass did not change the lifetimes and contribution of the kinetic components. This effect was ascribed to the decrease of conformational mobility of the protein domain between Y Z and P 680 , which resulted in the inhibition of Y Z  → P 680 + electron transfer in about half of the PS II population, wherein the recombination between Q A - and P 680 + occurred. The data indicate that PS II binds a larger number of water molecules as compared to PS I complexes. We conclude that our data disprove the "water replacement" hypothesis of trehalose matrix biopreservation., (Copyright © 2021 Elsevier B.V. All rights reserved.)

ESR, a light-driven proton pump from Exiguobacterium sibiricum, contains a lysine residue (Lys96) in the proton donor site. Substitution of Lys96 with a nonionizable residue greatly slows reprotonation of the retinal Schiff base. The recent study of electrogenicity of the K96A mutant revealed that overall efficiency of proton transport is decreased in the mutant due to back reactions (Siletsky et al., BBA, 2019). Similar to members of the proteorhodopsin and xanthorhodopsin families, in ESR the primary proton acceptor from the Schiff base, Asp85, closely interacts with His57. To examine the role of His57 in the efficiency of proton translocation by ESR, we studied the effects of H57N and H57N/K96A mutations on the pH dependence of light-induced pH changes in suspensions of Escherichia coli cells, kinetics of absorption changes and electrogenic proton transfer reactions during the photocycle. We found that at low pH (<5) the proton pumping efficiency of the H57N mutant in E. coli cells and its electrogenic efficiency in proteoliposomes is substantially higher than in the WT, suggesting that interaction of His57 with Asp85 sets the low pH limit for H + pumping in ESR. The electrogenic components that correspond to proton uptake were strongly accelerated at low pH in the mutant indicating that Lys96 functions as a very efficient proton donor at low pH. In the H57N/K96A mutant, a higher H + pumping efficiency compared with K96A was observed especially at high pH, apparently from eliminating back reactions between Asp85 and the Schiff base by the H57N mutation., (Copyright © 2020 Elsevier B.V. All rights reserved.)

The light-induced functioning of photosynthetic pigment-protein complex of photosystem II (PSII) is linked to the vectorial translocation of charges across the membrane, which results in the formation of voltage. Direct measurement of the light-induced voltage (∆V) generated by spinach oxygen-evolving PSII core complexes adsorbed onto a Millipore membrane filter (MF) on an indium tin oxide (ITO) electrode under continuous illumination has been performed. PSII was shown to participate in electron transfer from water to the ITO electrode, resulting in ∆V generation. No photovoltage was detected in PSII deprived of the water-oxidizing complex. The maximal and stable photoelectric signal was observed in the presence of disaccharide trehalose and 2,6-dichloro-1,4-benzoquinone, acting as a redox mediator between the primary quinone acceptor Q A of PSII and electrode surface. Long time preservation of the steady-state photoactivity at room temperature in a simple in design ITO|PSII-MF|ITO system may be related to the retention of water molecules attached to the PSII surface in the presence of trehalose.

The energy and charge-transfer processes in photosystem I (PS I) complexes isolated from cyanobacteria Thermosynechococcus elongatus and Synechocystis sp. PCC 6803 were investigated by pump-to-probe femtosecond spectroscopy. The formation of charge-transfer (CT) states in excitonically coupled chlorophyll a complexes (exciplexes) was monitored by measuring the electrochromic shift of β-carotene in the spectral range 500-510 nm. The excitation of high-energy chlorophyll in light-harvesting antenna of both species was not accompanied by immediate appearance of an electrochromic shift. In PS I from T. elongatus, the excitation of long-wavelength chlorophyll (LWC) caused a pronounced electrochromic effect at 502 nm assigned to the appearance of CT states of chlorophyll exciplexes. The formation of ion-radical pair P 700 + A 1 - at 40 ps was limited by energy transfer from LWC to the primary donor P 700 and accompanied by carotenoid bleach at 498 nm. In PS I from Synechocystis 6803, the excitation at 720 nm produced an immediate bidentate bleach at 690/704 nm and synchronous carotenoid response at 508 nm. The bidentate bleach was assigned to the formation of primary ion-radical state P B + Chl 2B - , where negative charge is localized predominantly at the accessory chlorophyll molecule in the branch B, Chl 2B . The following decrease of carotenoid signal at ~ 5 ps was ascribed to electron transfer to the more distant molecule Chl 3B . The reduction of phylloquinone in the sites A 1A and A 1B was accompanied by a synchronous blue-shift of the carotenoid response to 498 nm, pointing to fast redistribution of unpaired electron between two branches in favor of the state P B + A 1A - .

The Photosystem I (PSI) reaction center in cyanobacteria is comprised of ~96 chlorophyll (Chl) molecules, including six specialized Chl molecules denoted Chl1A/Chl1B (P 700 ), Chl2A/Chl2B, and Chl3A/Chl3B that are arranged in two branches and function in primary charge separation. It has recently been proposed that PSI from Chroococcidiopsis thermalis (Nürnberg et al. (2018) Science 360, 1210-1213) and Fischerella thermalis PCC 7521 (Hastings et al. (2019) Biochim. Biophys. Acta 1860, 452-460) contain Chl f in the positions Chl2A/Chl2B. We tested this proposal by exciting RCs from white-light grown (WL-PSI) and far-red light grown (FRL-PSI) F. thermalis PCC 7521 with femtosecond pulses and analyzing the optical dynamics. If Chl f were in the position Chl2A/Chl2B in FRL-PSI, excitation at 740 nm should have produced the charge-separated state P 700 + A 0 - followed by electron transfer to A 1 with a τ of ≤25 ps. Instead, it takes ~230 ps for the charge-separated state to develop because the excitation migrates uphill from Chl f in the antenna to the trapping center. Further, we observe a strong electrochromic shift at 685 nm in the final P 700 + A 1 - spectrum that can only be explained if Chl a is in the positions Chl2A/Chl2B. Similar arguments rule out the presence of Chl f in the positions Chl3A/Chl3B; hence, Chl f is likely to function solely as an antenna pigment in FRL-PSI. We additionally report the presence of an excitonically coupled homo- or heterodimer of Chl f absorbing around 790 nm that is kinetically independent of the Chl f population that absorbs around 740 nm., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Cyanobacterial photosystem I (PSI) functions as a light-driven cyt c 6 -ferredoxin/oxidoreductase located in the thylakoid membrane. In this work, the energy and charge transfer processes in PSI complexes isolated from Thermosynechococcus elongatus via conventional n-dodecyl-β-D-maltoside solubilization (DM-PSI) and a, to our knowledge, new detergent-free method using styrene-maleic acid copolymers (SMA-PSI) have been investigated by pump-to-probe femtosecond laser spectroscopy. In DM-PSI preparations excited at 740 nm, the excitation remained localized on the long-wavelength chlorophyll forms within 0.1-20 ps and revealed little or no charge separation and oxidation of the special pair, P700. The formation of ion-radical pair P700 + A1 - occurred with a characteristic time of 36 ps, being kinetically controlled by energy transfer from the long-wavelength chlorophyll to P 700 . Quite surprisingly, the detergent-free SMA-PSI complexes upon excitation by these long-wave pulses undergo an ultrafast (<100 fs) charge separation in ∼45% of particles. In the remaining complexes (∼55%), the energy transfer to P 700 occurred at ∼36 ps, similar to the DM-PSI. Both isolation methods result in a trimeric form of PSI, yet the SMA-PSI complexes display a heterogenous kinetic behavior. The much faster rate of charge separation suggests the existence of an ultrafast pathway for charge separation in the SMA-PSI that may be disrupted during detergent isolation., (Copyright © 2019 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Genomes of photoautotrophic organisms containing type I photosynthetic reaction center were searched for the rnf genes encoding Na+-translocating ferredoxin:NAD+ oxidoreductase (RNF). These genes were absent in heliobacteria, cyanobacteria, algae, and plants; however, genomes of many green sulfur bacteria (especially marine ones) were found to contain the full rnf operon. Analysis of RNA isolated from the marine green sulfur bacterium Chlorobium phaeovibrioides revealed a high level of rnf expression. It was found that the activity of Na+-dependent flavodoxin:NAD+ oxidoreductase detected in the membrane fraction of Chl. phaeovibrioides was absent in the membrane fraction of the freshwater green sulfur bacterium Chlorobaculum limnaeum, which is closely related to Chl. phaeovibrioides but whose genome lacks the rnf genes. Illumination of the membrane fraction of Chl. phaeovibrioides but not of Cba. limnaeum resulted in the light-induced NAD+ reduction. Based on the obtained data, we concluded that in some green sulfur bacteria, RNF may be involved in the NADH formation that should increase the efficiency of light energy conservation in these microorganisms and can serve as the first example of the use of Na+ energetics in photosynthetic electron transport chains.

Flavodoxins are small proteins with a non-covalently bound FMN that can accept two electrons and accordingly adopt three redox states: oxidized (quinone), one-electron reduced (semiquinone), and two-electron reduced (quinol). In iron-deficient cyanobacteria and algae, flavodoxin can substitute for ferredoxin as the electron carrier in the photosynthetic electron transport chain. Here, we demonstrate a similar function for flavodoxin from the green sulfur bacterium Chlorobium phaeovibrioides (cp-Fld). The expression of the cp-Fld gene, found in a close proximity with the genes for other proteins associated with iron transport and storage, increased in a low-iron medium. cp-Fld produced in Escherichia coli exhibited the optical, ERP, and electron-nuclear double resonance spectra that were similar to those of known flavodoxins. However, unlike all other flavodoxins, cp-Fld exhibited unprecedented stability of FMN semiquinone to oxidation by air and difference in midpoint redox potentials for the quinone-semiquinone and semiquinone-quinol couples (- 110 and - 530 mV, respectively). cp-Fld could be reduced by pyruvate:ferredoxin oxidoreductase found in the membrane-free extract of Chl. phaeovibrioides cells and photo-reduced by the photosynthetic reaction center found in membrane vesicles from these cells. The green sulfur bacterium Chl. phaeovibrioides appears thus to be a new type of the photosynthetic organisms that can use flavodoxin as an alternative electron carrier to cope with iron deficiency.

After removal of manganese ions responsible for light-driven water oxidation, redox-active tyrosine Y Z (tyrosine 161 of the D1 subunit) still remains the dominant electron donor to the photooxidized chlorophyll P 680 (P 680 + ) in the reaction center of photosystem 2 (PS2). Here, we investigated P 680 + reduction by Y Z under single-turnover flashes in Mn-depleted PS2 core complexes in the presence of weak acids and NH 4 Cl. Analysis of changes in the light-induced absorption at 830 nm (reflecting P 680 redox transitions) at pH 6.0 showed that P 680 + reduction is well approximated by two kinetic components with the characteristic times (τ) of ~7 and ~31 μs and relative contributions of ~54 and ~37%, respectively. In contrast to the very small effect of sodium formate (200 mM), addition of sodium acetate and NH 4 Cl increased the rate of electron transfer between Y Z and P 680 + approx. by a factor of 5. The suggestion that direct electron transfer from Y Z to P 680 + has a biphasic kinetics and reflects the presence of two different populations of PS2 centers was confirmed by the data obtained using direct electrometrical technique. It was demonstrated that the submillisecond two-phase kinetics of the additional electrogenic phase in the kinetics of photoelectric response due to the electron transfer between Y Z and P 680 + is significantly accelerated in the presence of acetate or ammonia. These results contribute to the understanding of the mechanism of interaction between the oxidized tyrosine Y Z and exogenous substances (including synthetic manganese-containing compounds) capable of photooxidation of water molecule in the manganese-depleted PS2 complexes.

In the present study, we have investigated the effect of hydroxyectoine (Ect-OH), a heterocyclic amino acid, on oxygen evolution in photosystem II (PS II) membrane fragments and on photoinhibition of Mn-depleted PS II (apo-WOC-PS II) preparations. The degree of photoinhibition of apo-WOC-PS II preparations was estimated by the loss of the capability of exogenous electron donor (sodium ascorbate) to restore the amplitude of light-induced changes of chlorophyll fluorescence yield (∆F). It was found that Ect-OH (i) stimulates the oxygen-evolving activity of PS II, (ii) accelerates the electron transfer from exogenous electron donors (K 4 [Fe(CN) 6 ], DPC, TMPD, Fe 2+ , and Mn 2+ ) to the reaction center of apo-WOC-PS II, (iii) enhances the protective effect of exogenous electron donors against donor-side photoinhibition of apo-WOC-PS II preparations. It is assumed that Ect-OH can serve as an artificial electron donor for apo-WOC-PS II, which does not directly interact with either the donor or acceptor side of the reaction center. We suggest that the protein conformation in the presence of Ect-OH, which affects the extent of hydration, becomes favorable for accepting electrons from exogenous donors. To our knowledge, this is the first study dealing with redox activity of Ect-OH towards photosynthetic pigment-protein complexes.

The dependence on temperature of tryptophan fluorescence lifetime in trimeric photosystem I (PSI) complexes from cyanobacteria Synechocystis sp. PCC 6803 during the heating of pre-frozen to - 180 °C in the dark or in the light-activated preparations has been studied. Fluorescence lifetime in samples frozen in the light was longer than in samples frozen in the dark. For samples in 65% glycerol at λ reg  = 335 nm and at 20 °C, the lifetime of components were as follows: τ 1  ≈ 1.2 ns, τ 2  ≈ 4.9 ns, and τ 3  ≈ 20 ns. The contribution of the first component was negligible. To analyze the contribution of components 2 and 3 derived from frozen-thawed samples, two temperature ranges from - 180 to - 90 °C and above - 90 °C are considered. In doing so, the contributions of these components appear antiphase course to each other. The dependence on temperature of these contributions is explained by the influence of the microconformational protein dynamics on the tryptophan fluorescence lifetime. In the present work, a comparative analysis of temperature-dependent conformational dynamics and electron transfer in cyanobacterial PSI (Schlodder et al., in Biochemistry 37:9466-9476, 1998) and Rhodobacter sphaeroides reaction center complexes (Knox et al., in J Photochem Photobiol B 180:140-148, 2018) was also carried out.

ESR from Exiguobacterium sibiricum is a retinal protein which functions as a proton pump. Unusual feature of ESR is that a lysine residue is present at a site for the internal proton donor, which in other proton pumps is a carboxylic residue. Replacement of Lys96 with alanine slows reprotonation of the Schiff base by two orders of magnitude, indicating that Lys96 and interacting water molecules function as internal proton donor to the Schiff base. In this work we examined time resolved generation of light-induced electric potential ΔΨ by the K96A mutant reconstituted into proteoliposomes. We found that the ΔΨ component, which accompanied reprotonation of the Schiff base in wild type ESR, was not only slowed but also decreased greatly in the mutant, and negative phase appeared at high pH. This indicates a higher probability of back reactions in ESR than in bacteriorhodopsin since no negative components have been observed in homologous mutants of BR, D96N and D96A. The higher rate of back reactions in ESR is probably caused by different arrangement of the proton acceptor site compared to that in BR and different sequence of proton release and uptake. Addition of sodium azide, which substitutes for the internal proton donor, restores both the rate and amplitude of the ΔΨ components related to the Schiff base reprotonation in the K96A mutant. This indicates that overall proton transport results from competition of forward and reverse reactions, and emphasizes the importance of internal donor for high efficiency and directionality of H + transfer., (Copyright © 2018 Elsevier B.V. All rights reserved.)

An electrometrical technique was used to investigate electron transfer between synthetic binuclear manganese (Mn) complexes, designated M - 2 and M - 3, and the redox-active neutral tyrosine radical (Y Z • ) in proteoliposomes containing Mn-depleted photosystem II (PS II) core particles in response to single laser flashes. In the absence of Mn-containing compounds, the observed flash-induced membrane potential (ΔΨ) decay was mainly due to charge recombination between the reduced primary quinone acceptor Q A - and the oxidized Y Z • . More significant slowing down of the ΔΨ decay in the presence of lower concentrations of M - 2 and M - 3 associated with electron donation from Mn in the Mn-binding site to Y Z • indicates that these synthetic compounds are more effective electron donors than MnCl 2 . The exponential fitting of the kinetics of additional electrogenic components of ΔΨ rise in the presence of Mn-containing compounds revealed the following relative amplitudes (A) and lifetimes (τ): for MnCl 2 - A∼ 3.5, τ∼150 μs, for M - 2 - A∼5%, τ∼1.4 ms, and for M - 3 - A∼5.5%, τ∼150 μs. This suggests that the efficiency of the manganese complexes in electron donation depends on the chemical nature of ligands. The experiments with EDTA-treated samples indicated that the ligands for M - 2 and M - 3 are required for their tight binding with the PS II reaction center. The obtained results demonstrate the importance of understanding the molecular mechanism(s) of flash-induced electrogenic reduction of the tyrosine radical Y Z • by synthetic Mn complexes capable of splitting water into oxygen and reducing equivalents., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Bacterial Na + -transporting rhodopsins convert solar energy into transmembrane ion potential difference. Typically, they are strictly specific for Na + , but some can additionally transport H + . To determine the structural basis of cation promiscuity in Na + -rhodopsins, we compared their primary structures and found a single position that harbors a cysteine in strictly specific Na + -rhodopsins and a serine in the promiscuous Krokinobacter eikastus Na + -rhodopsin (Kr2). A Cys253Ser variant of the strictly specific Dokdonia sp. PRO95 Na + -rhodopsin (NaR) was indeed found to transport both Na + and H + in a light-dependent manner when expressed in retinal-producing Escherichia coli cells. The dual specificity of the NaR variant was confirmed by analysis of its photocycle, which revealed an acceleration of the cation-capture step by comparison with the wild-type NaR in a Na + -deficient medium. The structural basis for the dependence of the Na + /H + specificity in Na + -rhodopsin on residue 253 remains to be determined., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Light-driven H + , Cl - and Na + rhodopsin pumps all use a covalently bound retinal molecule to capture light energy. Some H + -pumping rhodopsins (xanthorhodopsins; XRs) additionally contain a carotenoid antenna for light absorption. Comparison of the available primary and tertiary structures of rhodopsins pinpointed a single Thr residue (Thr216) that presumably prevents carotenoid binding to Na + -pumping rhodopsins (NaRs). We replaced this residue in Dokdonia sp. PRO95 NaR with Gly, which is found in the corresponding position in XRs, and produced a variant rhodopsin in a ketocarotenoid-synthesising Escherichia coli strain. Unlike wild-type NaR, the isolated variant protein contained the tightly bound carotenoids canthaxanthin and echinenone. These carotenoids were visible in the absorption, circular dichroism and fluorescence excitation spectra of the Thr216Gly-substituted NaR, which indicates their function as a light-harvesting antenna. The amino acid substitution and the bound carotenoids did not affect the NaR photocycle. Our findings suggest that the antenna function was recently lost during NaR evolution but can be easily restored by site-directed mutagenesis.

Photosystem I (PSI) is a Type-I reaction center and is the largest photosynthetic complex to be characterized. In cyanobacteria, PSI is organized as a trimer with a three-fold axis of symmetry. Recently, a tetrameric form of PSI has been identified in cyanobacteria. Plastids in plants and algae only contain monomeric PSI, suggesting that tetrameric PSI may be key in the transition from ancestral cyanobacterial trimeric PSI to plant/algal monomeric PSI. We have investigated the kinetics of electron transfer to the initial acceptor in PSI tetramer isolated from Chroococcidiopsis TS-821. Using a pump-probe technique with 25 fs low-energy, 720 nm pump pulses, we measure the ultrafast (<100 fs) conversion of a delocalized exciton into a charge-separated state between the primary donor P 700 and the primary acceptor A 0 . Comparison with previous pump-probe analysis of the trimeric PSI complexes from Synechocystis sp PCC 6803 (Shelaev et al. [2010] Biochim Biophys Acta, 1797, 1410-1420) reveals that the tetrameric (PSI) complexes from Chroococcidiopsis sp TS-821 are quite similar. The transfer of an electron from the A 0 to the following acceptor A1 (phylloquinone) takes place in a time frame of about 30 ps, which is slightly longer compared to PSI trimeric complex (~24 ps). The slight spectral differences between trimeric and tetrameric PSI complexes are discussed., (© 2018 The American Society of Photobiology.)

Lumenal extrinsic proteins PsbO, PsbP, and PsbQ of photosystem II (PSII) protect the catalytic cluster Mn 4 CaO 5 of oxygen-evolving complex (OEC) from the bulk solution and from soluble compounds in the surrounding medium. Extraction of PsbP and PsbQ proteins by NaCl-washing together with chelator EGTA is followed also by the depletion of Ca 2+ cation from OEC. In this study, the effects of PsbP and PsbQ proteins, as well as Ca 2+ extraction from OEC on the kinetics of the reduced primary electron acceptor (Q A - ) oxidation, have been studied by fluorescence decay kinetics measurements in PSII membrane fragments. We found that in addition to the impairment of OEC, removal of PsbP and PsbQ significantly slows the rate of electron transfer from Q A - to the secondary quinone acceptor Q B . Electron transfer from Q A - to Q B in photosystem II membranes with an occupied Q B site was slowed down by a factor of 8. However, addition of EGTA or CaCl 2 to NaCl-washed PSII did not change the kinetics of fluorescence decay. Moreover, the kinetics of Q A - oxidation by Q B in Ca-depleted PSII membranes obtained by treatment with citrate buffer at pH 3.0 (such treatment keeps all extrinsic proteins in PSII but extracts Ca 2+ from OEC) was not changed. The results obtained indicate that the effect of NaCl-washing on the Q A - to Q B electron transport is due to PsbP and PsbQ extrinsic proteins extraction, but not due to Ca 2+ depletion.

The ultrafast primary charge separation in Photosystem I (PS I) excited by femtosecond pulses centered at 720 and 760nm was studied by pump-to-probe laser spectroscopy. The absorbance in the red edge of PS I absorption spectrum has an unusual exponential dependence on wavelength. The cutoff of short wavelength components of 760nm pulse allows direct excitation of reaction center chlorophyll molecules without involvement of light-harvesting antenna. The transient spectrum manifests the features of the primary ion-radical pair P 700 + A 0 - at time delay <180fs, followed by formation of the secondary pair P 700 + A 1 - with a characteristic time of 26ps. The obtained data are rationalized in the framework of adiabatic three-state model that includes the chlorophyll dimer P 700 and two symmetrically arranged nearest chlorophyll molecules of A 0 . The arrangement of chlorophylls results in strong electronic coupling between P 700 and A 0 . Excitation in the maximum of P 700 absorption generates electronic states with the highest contribution from P 700 *, whereas excitation in the far-red edge predominantly generates charge transfer state P 700 + A 0 - in both branches of redox-cofactors. The three-level model accounts for a flat-bottomed potential surface of the excited state and adiabatic character of electron transfer between P 700 and A 0 , providing a microscopic explanation of the ultrafast formation of P 700 + A 0 - and exponential decline of PS I absorption., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Interaction of photosystem I (PS I) complexes from cyanobacteria Synechocystis sp. PCC 6803 containing various quinones in the A 1 -site (phylloquinone PhQ in the wild-type strain (WT), and plastoquinone PQ or 2,3-dichloronaphthoquinone Cl 2 NQ in the menB deletion strain) and different numbers of Fe 4 S 4 clusters (intact WT and F X -core complexes depleted of F A /F B centers) with external acceptors has been studied. The efficiency of interaction was estimated by measuring the light-induced absorption changes at 820 nm due to the reduction of the special pair of chlorophylls (P 700 + ) by an external acceptor(s). It was shown that externally added Cl 2 NQ is able to effectively accept electrons from the terminal iron-sulfur clusters of PS I. Moreover, the efficiency of Cl 2 NQ as external acceptor was higher than the efficiency of the commonly used artificial electron acceptor, methylviologen (MV) for both the intact WT PS I and for the F X -core complexes. The comparison of the efficiency of MV interaction with different types of PS I complexes revealed gradual decrease in the following order: intact WT > menB > F X -core. The effect of MV on the recombination kinetics in menB complexes of PS I with Cl 2 NQ in the A 1 -site differed significantly from all other PS I samples. The obtained effects are considered in terms of kinetic efficiency of electron acceptors in relation to thermodynamic and structural characteristics of PS I complexes.

In the thylakoid membrane of green plants, cyanobacteria and algae, photosystem II (PSII) uses light energy to split water and generate molecular oxygen. In the opposite process of the biochemical transformation of dioxygen, in heterotrophs, the terminal respiratory oxidases (TRO) are at the end of the respiratory chain in mitochondria and in plasma membrane of many aerobic bacteria reducing dioxygen back to water. Despite the different sources of free energy (light or oxidation of the substrates), energy conversion by these enzymes is based on the spatial organization of enzymatic reactions in which the conversion of water to dioxygen (and vice versa) involves the transfer of protons and electrons in opposite directions across the membrane, which is accompanied by generation of proton-motive force. Similar and distinctive features in structure and function of these important energy-converting molecular machines are described. Information about many fascinating parallels between the mechanisms of TRO and PSII could be used in the artificial light-driven water-splitting process and elucidation of energy conversion mechanism in protein pumps.

In a direct experiment, the rate constants of photochemical k p and non-photochemical k p + quenching of the chlorophyll fluorescence have been determined in spinach photosystem II (PS II) membrane fragments, oxygen-evolving PS II core, as well as manganese-depleted PS II particles using pulse fluorimetry. In the dark-adapted reaction center(s) (RC), the fluorescence decay kinetics of the antenna were measured at low-intensity picosecond pulsed excitation. To create a "closed" P680 + Q A - state, RCs were illuminated by high-intensity actinic flash 8 ns prior to the measuring flash. The obtained data were approximated by the sum of two decaying exponents. It was found that the antennae fluorescence quenching efficiency by the oxidized photoactive pigment of RC P680 + was about 1.5 times higher than that of the neutral P680 state. These results were confirmed by a single-photon counting technique, which allowed to resolve the additional slow component of the fluorescence decay. Slow component was assigned to the charge recombination of P680 + Pheo - in PS II RC. Thus, for the first time, the ratio k p + /k p  ≅ 1.5 was found directly. The mechanism of the higher efficiency of non-photochemical quenching comparing to photochemical quenching is discussed.

A retinal protein from Exiguobacterium sibiricum (ESR) functions as a light-driven proton pump. Unlike other proton pumps, it contains Lys96 instead of a usual carboxylic residue in the internal proton donor site. Nevertheless, the reprotonation of the Schiff base occurs fast, indicating that Lys96 facilitates proton transfer from the bulk. In this study we examined kinetics of light-induced transmembrane electrical potential difference, ΔΨ, generated in proteoliposomes reconstituted with ESR. We show that total magnitude of ΔΨ is comparable to that produced by bacteriorhodopsin but its kinetic components and their pH dependence are substantially different. The results are in agreement with the earlier finding that proton uptake precedes reprotonation of the Schiff base in ESR, suggesting that Lys96 is unprotonated in the initial state and gains a proton transiently in the photocycle. The electrogenic phases and the photocycle transitions related to proton transfer from the bulk to the Schiff base are pH dependent. At neutral pH, they occur with τ 0.5ms and 4.5ms. At alkaline pH, the fast component ceases and Schiff base reprotonation slows. At pH8.4, a spectrally silent electrogenic component with τ 0.25ms is detected, which can be attributed to proton transfer from the bulk to Lys96. At pH5.1, the amplitude of ΔΨ decreases 10 fold, reflecting a decreased yield and rate of proton transfer, apparently from protonation of the acceptor (Asp85-His57 pair) in the initial state. The features of the photoelectric potential generation correlate with the ESR structure and proposed mechanism of proton transfer., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Recently, it has been shown that the addition of 1M trehalose leads to the increase of the rate of oxygen photoconsumption associated with activation of electron transport in the reaction center of photosystem 2 (PS2) in Mn-depleted PS2 membranes (apo-WOC-PS2) [37]. In the present work the effect of trehalose on photoinhibition of apo-WOC-PS2 preparations (which are characterized by a high sensitivity to the donor side photoinhibition of PS2) was investigated. The degree of photoinhibition was estimated by the loss of the capability of exogenous electron donor (sodium ascorbate) to reactivate the electron transport (measured by light-induced changes of chlorophyll fluorescence yield (∆F)) in apo-WOC-PS2. It was found that 1M trehalose enhanced the Mn 2+ -dependent suppression of photoinhibition of apo-WOC-PS2: in the presence of trehalose the addition of 0.2μM Mn 2+ (corresponding to 2 Mn 2+ per one reaction center) was sufficient for an almost complete suppression of the donor side photoinhibition of the complex. In the absence of trehalose it was necessary to add 100μM Mn 2+ to achieve a similar result. The effect of trehalose was observed during photoinhibition of apo-WOC-PS2 at low (15μmolphotons -1 m -2 ) and high (200μmolphotons -1 m -2 ) light intensity. When Mn 2+ was replaced by other PS2 electron donors (ferrocyanide, DPC) as well as by Ca 2+ the protective effect of trehalose was not observed. It was also found that 1M trehalose decreased photoinhibition of apo-WOC-PS2 if the samples contained endogenous manganese (1-2 Mn ions per one RC was enough for the maximum protection effect). It is concluded that structural changes in PS2 caused by the addition of trehalose enhance the capability of photochemical reaction centers of apo-WOC-PS2 to accept electrons from manganese (both exogenous and endogenous), which in turn leads to a considerable suppression of the donor side photoinhibition of PS2., (Copyright © 2016 Elsevier B.V. All rights reserved.)

In direct experiments, rate constants of photochemical (kP) and non-photochemical (kP(+)) fluorescence quenching were determined in membrane fragments of photosystem II (PSII), in oxygen-evolving PSII core particles, as well as in core particles deprived of the oxygen-evolving complex. For this purpose, a new approach to the pulse fluorometry method was implemented. In the "dark" reaction center (RC) state, antenna fluorescence decay kinetics were measured under low-intensity excitation (532 nm, pulse repetition rate 1 Hz), and the emission was registered by a streak camera. To create a "closed" [P680(+)QA(-)] RC state, a high-intensity pre-excitation pulse (pump pulse, 532 nm) of the sample was used. The time advance of the pump pulse against the measuring pulse was 8 ns. In this experimental configuration, under the pump pulse, the [P680(+)QA(-)] state was formed in RC, whereupon antenna fluorescence kinetics was measured using a weak testing picosecond pulsed excitation light applied to the sample 8 ns after the pump pulse. The data were fitted by a two-exponential approximation. Efficiency of antenna fluorescence quenching by the photoactive RC pigment in its oxidized (P680(+)) state was found to be ~1.5 times higher than that of the neutral (P680) RC state. To verify the data obtained with a streak camera, control measurements of PSII complex fluorescence decay kinetics by the single-photon counting technique were carried out. The results support the conclusions drawn from the measurements registered with the streak camera. In this case, the fitting of fluorescence kinetics was performed in three-exponential approximation, using the value of τ1 obtained by analyzing data registered by the streak camera. An additional third component obtained by modeling the data of single photon counting describes the P680(+)Pheo(-) charge recombination. Thus, for the first time the ratio of kP(+)/kP = 1.5 was determined in a direct experiment. The mechanisms of higher efficiency for non-photochemical antenna fluorescence quenching by RC cation radical in comparison to that of photochemical quenching are discussed.

Discovery of the light-driven sodium-motive pump Na(+)-rhodopsin (NaR) has initiated studies of the molecular mechanism of this novel membrane-linked energy transducer. In this paper, we investigated the photocycle of NaR from the marine flavobacterium Dokdonia sp. PRO95 and identified electrogenic and Na(+)-dependent steps of this cycle. We found that the NaR photocycle is composed of at least four steps: NaR519 + hv → K585 → (L450↔M495) → O585 → NaR519. The third step is the only step that depends on the Na(+) concentration inside right-side-out NaR-containing proteoliposomes, indicating that this step is coupled with Na(+) binding to NaR. For steps 2, 3, and 4, the values of the rate constants are 4×10(4) s(-1), 4.7 × 10(3) M(-1) s(-1), and 150 s(-1), respectively. These steps contributed 15, 15, and 70% of the total membrane electric potential (Δψ ~ 200 mV) generated by a single turnover of NaR incorporated into liposomes and attached to phospholipid-impregnated collodion film. On the basis of these observations, a mechanism of light-driven Na(+) pumping by NaR is suggested.

It is known that the removal of manganese from the water-oxidizing complex (WOC) of photosystem 2 (PS2) leads to activation of oxygen photoconsumption (OPC) [Khorobrykh et al., 2002; Yanykin et al., 2010] that is accompanied by the formation of organic hydroperoxides on the electron-donor side of PS2 [Khorobrykh et al., 2011]. In the present work the effect of trehalose on the OPC in Mn-depleted PS2 preparations (apo-WOC-PS2) was investigated. A more than two-fold increase of the OPC is revealed upon the addition of 1M trehalose. Drastic (30%-70%) inhibition of the OPC upon the addition of either electron acceptor or electron donor indicates that the trehalose-induced activation of the OPC occurs on both donor and acceptor sides of PS2. A two-fold increase in the rate of superoxide-anion radical photoproduction on the electron-acceptor side of PS2 was also shown. Applying the "variable" chlorophyll fluorescence (ΔF) it was shown that the addition of trehalose induces: (i) a significant increase in the ability of exogenous Mn(2+) to donate electrons to the reaction center of PS2, (ii) slowing down the photoaccumulation of the primary quinone electron acceptor of PS2 (QA(-)) under aerobic conditions, (iii) acceleration of the reoxidation of QA(-) by QB (and by QB(-)) as well as the replacement of QB(2-) by a fully oxidized plastoquinone, and (iv) restoration of the electron transfer between the quinone electron carriers in the so-called "closed reaction centers of PS2" (their content in the apo-WOC-PS2 is 41%). It is suggested that the trehalose-induced increase in efficiency of the O2 interaction with the electron-donor and electron-acceptor sides of apo-WOC-PS2 is due to structural changes leading to both a decrease in the proportion of the "closed PS2 reaction centers" and an increase in the electron transfer rate in PS2., (Copyright © 2015 Elsevier B.V. All rights reserved.)

The pigment-protein complex of photosystem I (PS I) catalyzes light-driven oxidation of plastocyanin or cytochrome c6 and reduction of ferredoxin or flavodoxin in oxygenic photosynthetic organisms. In this review, we describe the current state of knowledge of the processes of excitation energy transfer and formation of the primary and secondary ion-radical pairs within PS I. The electron transfer reaction involving quinone cofactor in the A1 site and its role in providing asymmetry of electron transport as well as interaction with oxygen and ascorbate in PS I are discussed.

The pigment-protein complex of photosystem 2 (PS 2) catalyzes the light-driven oxidation of water molecule and the reduction of plastoquinone. In this work, we studied the effect of the disaccharide trehalose, which is unique in its physicochemical properties, on isolated PS 2 complex. It was found that trehalose significantly stimulated the steady-state rate of oxygen evolution. The study of single flash-induced fluorescence decay kinetics demonstrated that trehalose did not affect the rate of QA(-) oxidation, although it led to an increase in the relative fractions of PS 2 reaction centers capable of QA(-) oxidation. Trehalose also prevented PS 2 complexes from being inactivated on prolonged storage. We propose that in the presence of trehalose, which affects the extent of hydration, the protein can preferentially exist in a more optimal conformation for effective functioning.

O2 reduction was investigated in photosystem I (PSI) complexes isolated from cyanobacteria Synechocystis sp. PCC 6803 wild type (WT) and menB mutant strain, which is unable to synthesize phylloquinone and contains plastoquinone at the quinone-binding site A1. PSI complexes from WT and menB mutant exhibited different dependencies of O2 reduction on light intensity, namely, the values of O2 reduction rate in WT did not reach saturation at high intensities, in contrast to the values in menB mutant. The obtained results suggest the immediate phylloquinone involvement in the light-induced O2 reduction by PSI., (Copyright © 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)

Ascorbate is one of the key participants of the antioxidant defense in plants. In this work, we have investigated the interaction of ascorbate with the chloroplast electron transport chain and isolated photosystem I (PSI), using the EPR method for monitoring the oxidized centers [Formula: see text] and ascorbate free radicals. Inhibitor analysis of the light-induced redox transients of P700 in spinach thylakoids has demonstrated that ascorbate efficiently donates electrons to [Formula: see text] via plastocyanin. Inhibitors (DCMU and stigmatellin), which block electron transport between photosystem II and Pc, did not disturb the ascorbate capacity for electron donation to [Formula: see text]. Otherwise, inactivation of Pc with CN(-) ions inhibited electron flow from ascorbate to [Formula: see text]. This proves that the main route of electron flow from ascorbate to [Formula: see text] runs through Pc, bypassing the plastoquinone (PQ) pool and the cytochrome b 6 f complex. In contrast to Pc-mediated pathway, direct donation of electrons from ascorbate to [Formula: see text] is a rather slow process. Oxidized ascorbate species act as alternative oxidants for PSI, which intercept electrons directly from the terminal electron acceptors of PSI, thereby stimulating photooxidation of P700. We investigated the interaction of ascorbate with PSI complexes isolated from the wild type cells and the MenB deletion strain of cyanobacterium Synechocystis sp. PCC 6803. In the MenB mutant, PSI contains PQ in the quinone-binding A1-site, which can be substituted by high-potential electron carrier 2,3-dichloro-1,4-naphthoquinone (Cl2NQ). In PSI from the MenB mutant with Cl2NQ in the A1-site, the outflow of electrons from PSI is impeded due to the uphill electron transfer from A1 to the iron-sulfur cluster FX and further to the terminal clusters FA/FB, which manifests itself as a decrease in a steady-state level of [Formula: see text]. The addition of ascorbate promoted photooxidation of P700 due to stimulation of electron outflow from PSI to oxidized ascorbate species. Thus, accepting electrons from PSI and donating them to [Formula: see text], ascorbate can mediate cyclic electron transport around PSI. The physiological significance of ascorbate-mediated electron transport is discussed.