Multimodal non-invasive probing of stress-induced carotenogenesis in the cells of microalga Bracteacoccus aggregatus.

Microalgae are the richest source of natural carotenoids-accessory photosynthetic pigments used as natural antioxidants, safe colorants, and nutraceuticals. Microalga Bracteacoccus aggregatus IPPAS C-2045 responds to stresses, including high light, with carotenogenesis-gross accumulation of secondary carotenoids (the carotenoids structurally and energetically uncoupled from photosynthesis). Precise mechanisms of cytoplasmic transport and subcellular distribution of the secondary carotenoids under stress are still unknown. Using multimodal imaging combining micro-Raman imaging (MRI), fluorescent lifetime (τ) imaging (FLIM), and transmission electron microscopy (TEM), we monitored ultrastructural and biochemical rearrangements of B. aggregatus cells during the stress-induced carotenogenesis. MRI revealed a decline in the diversity of molecular surrounding of the carotenoids in the cells compatible with the relocation of the bulk of the carotenoids in the cell from functionally and structurally heterogeneous photosynthetic apparatus to the more homogenous lipid matrix of the oleosomes. Two-photon FLIM highlighted the pigment transformation in the cell during the stress-induced carotenogenesis. The structures co-localized with the carotenoids with shorter τ (mainly chloroplast) shrunk, whereas the structures harboring secondary carotenoids with longer τ (mainly oleosomes) expanded. These changes were in line with the ultrastructural data (TEM). Fluorescence of B. aggregatus carotenoids, either in situ or in acetone extracts, possessed a surprisingly long lifetime. We hypothesize that the extension of τ of the carotenoids is due to their aggregation and/or association with lipids and proteins. The propagation of the carotenoids with prolonged τ is considered to be a manifestation of the secondary carotenogenesis suitable for its non-invasive monitoring with multimodal imaging.

Femtosecond optical studies of the primary charge separation reactions in far-red photosystem II from Synechococcus sp. PCC 7335.

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 Qy 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 ChlD1 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 PD1+ChlD1- 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 PheoD1 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, PD1+ChlD1- can be partially formed from the excited (PD1ChlD1)* state.

Upright proton therapy for esthesioneuroblastoma: a single-institution experience.

Aim: This study presents an analysis (efficacy and toxicity) of outcomes in patients with esthesioneuroblastoma after pencil beam proton therapy with a fixed beamline in the upright position. Background: Esthesioneuroblastoma (ENB) is an extremely rare tumor of sinonasal area located in critical proximity to vital structures. Proton therapy (PT) is often considered the optimal radiation treatment for head-and-neck tumors, although of limited availability. Upright PT delivered using fixed pencil beamline and rotating chair is a fairly promising option. Methods: This is a single-center experience describing the outcomes of PT in 14 patients with ENB treated between January 2016 and October 2022; half of the cases had a history of previous irradiation. The therapy was applied using a fixed pencil beamline with 6D-chair for positioning. The median dose was 63 GyRBE (total range 48-70 GyRBE; based on 1.1 RBE multiplier for protons) with 2.0 GyRBE per fraction. The mean gross tumor volume was 109.5 cm3 (17.1-257.7 cm3). Patient demography, pathology, treatment parameters and toxicity data were analyzed. Radiation-induced reactions were assessed according to the Common Terminology Criteria for Adverse Events (CTCAE) v 4.0. Results: The median follow-up time was 28 months. The 1- and 2-year locoregional control rates constituted 100% and 88.9%, respectively; the median duration of local control was 52 months. The 1- and 2-year progression-free survival (PFS) rates constituted 92.9% and 75.0%, respectively; the median PFS duration was 52 months. The 1- and 2-year overall survival (OS) rates constituted 92.9% and 84.4%, respectively. Two patients died of non-cancer-related causes (coronavirus-induced pneumonia) and 1 patient died of tumor progression. All patients tolerated PT well without any treatment gaps. Serious late toxicity reactions included glaucoma in 1 patient and cataract in 2 patients, in over half a year since irradiation. Conclusion: PT with upright design of the unit affords promising outcomes in terms of disease control and toxicity rates in ENB, a sinonasal tumor of complicated localization.

Inverted region in the reaction of the quinone reduction in the A1-site of photosystem I from cyanobacteria.

Photosystem I from the menB strain of Synechocystis sp. PCC 6803 containing foreign quinones in the A1 sites was used for studying the primary steps of electron transfer by pump-probe femtosecond laser spectroscopy. The free energy gap (- ΔG) of electron transfer between the reduced primary acceptor A0 and the quinones bound in the A1 site varied from 0.12 eV for the low-potential 1,2-diamino-anthraquinone to 0.88 eV for the high-potential 2,3-dichloro-1,4-naphthoquinone, compared to 0.5 eV for the native phylloquinone. It was shown that the kinetics of charge separation between the special pair chlorophyll P700 and the primary acceptor A0 was not affected by quinone substitutions, whereas the rate of A0 → A1 electron transfer was sensitive to the redox-potential of quinones: the decrease of - ΔG by 400 meV compared to the native phylloquinone resulted in a ~ fivefold slowing of the reaction The presence of the asymmetric inverted region in the ΔG dependence of the reaction rate indicates that the electron transfer in photosystem I is controlled by nuclear tunneling and should be treated in terms of quantum electron-phonon interactions. A three-mode implementation of the multiphonon model, which includes modes around 240 cm-1 (large-scale protein vibrations), 930 cm-1 (out-of-plane bending of macrocycles and protein backbone vibrations), and 1600 cm-1 (double bonds vibrations) was applied to rationalize the observed dependence. The modes with a frequency of at least 1600 cm-1 make the predominant contribution to the reorganization energy, while the contribution of the "classical" low-frequency modes is only 4%.

Lel A. Drachev and the Direct Electrometric Method.

In the bioenergetics studies, the direct electrometric method played an important role. This method is based on measuring the electrical potential difference (Δψ) between two compartments of the experimental cell generated by some membrane proteins. These proteins are incorporated into closed lipid-protein membrane vesicles associated with an artificial lipid membrane that separates the compartments. The very existence of such proteins able to generate Δψ was one of the consequences of Peter Mitchell's chemiosmotic concept. The discovery and investigation of their functioning contributed to the recognition of this concept and, eventually the well-deserved awarding of the Nobel Prize to P. Mitchell. Lel A. Drachev (1926-2022) was one of the main authors of the direct electrometrical method. With his participation, key studies were carried out on the electrogenesis of photosynthetic and respiratory membrane proteins, including bacteriorhodopsin, visual rhodopsin, photosynthetic bacterial reaction centers, cytochrome oxidase and others.

Electrometric and Electron Paramagnetic Resonance Measurements of a Difference in the Transmembrane Electrochemical Potential: Photosynthetic Subcellular Structures and Isolated Pigment-Protein Complexes.

A transmembrane difference in the electrochemical potentials of protons (ΔµH+) serves as a free energy intermediate in energy-transducing organelles of the living cell. The contributions of two components of the ΔµH+ (electrical, Δψ, and concentrational, ΔpH) to the overall ΔµH+ value depend on the nature and lipid composition of the energy-coupling membrane. In this review, we briefly consider several of the most common instrumental (electrometric and EPR) methods for numerical estimations of Δψ and ΔpH. In particular, the kinetics of the flash-induced electrometrical measurements of Δψ in bacterial chromatophores, isolated bacterial reaction centers, and Photosystems I and II of the oxygenic photosynthesis, as well as the use of pH-sensitive molecular indicators and kinetic data regarding pH-dependent electron transport in chloroplasts, have been reviewed. Further perspectives on the application of these methods to solve some fundamental and practical problems of membrane bioenergetics are discussed.

Role of pheophytin a in the primary charge separation of photosystem I from Acaryochloris marina: Femtosecond optical studies of excitation energy and electron transfer reactions.

Photosystem I (PSI) of the cyanobacterium Acaryochloris marina is capable of performing an efficient photoelectrochemical conversion of far-red light due to its unique suite of cofactors. Chlorophyll d (Chl-d) has been long known as the major antenna pigment in the PSI from A. marina, while the exact cofactor composition of the reaction centre (RC) was established only recently by cryo-electron microscopy. The RC consists of four Chl-d molecules, and, surprisingly, two molecules of pheophytin a (Pheo-a), which provide a unique opportunity to resolve, spectrally and kinetically, the primary electron transfer reactions. Femtosecond transient absorption spectroscopy was here employed to observe absorption changes in the 400-860 nm spectral window occurring in the 0.1-500 ps timescale upon unselective antenna excitation and selective excitation of the Chl-d special pair P740 in the RC. A numerical decomposition of the absorption changes, including principal component analysis, allowed the identification of P740(+)Chld2(-) as the primary charge separated state and P740(+)Pheoa3(-) as the successive, secondary, radical pair. A remarkable feature of the electron transfer reaction between Chld2 and Pheoa3 is the fast, kinetically unresolved, equilibrium with an estimated ratio of 1:3. The energy level of the stabilised ion-radical state P740(+)Pheoa3(-) was determined to be ~60 meV below that of the RC excited state. In this regard, the energetics and the structural implications of the presence of Pheo-a in the electron transfer chain of PSI from A. marina are discussed, also in comparison with those of the most diffused Chl-a binding RC.

2D-GaN/AlN Multiple Quantum Disks/Quantum Well Heterostructures for High-Power Electron-Beam Pumped UVC Emitters.

This article describes GaN/AlN heterostructures for ultraviolet-C (UVC) emitters with multiple (up to 400 periods) two-dimensional (2D)-quantum disk/quantum well structures with the same GaN nominal thicknesses of 1.5 and 16 ML-thick AlN barrier layers, which were grown by plasma-assisted molecular-beam epitaxy in a wide range of gallium and activated nitrogen flux ratios (Ga/N2*) on c-sapphire substrates. An increase in the Ga/N2* ratio from 1.1 to 2.2 made it possible to change the 2D-topography of the structures due to a transition from the mixed spiral and 2D-nucleation growth to a purely spiral growth. As a result, the emission energy (wavelength) could be varied from 5.21 eV (238 nm) to 4.68 eV (265 nm) owing to the correspondingly increased carrier localization energy. Using electron-beam pumping with a maximum pulse current of 2 A at an electron energy of 12.5 keV, a maximum output optical power of 50 W was achieved for the 265 nm structure, while the structure emitting at 238 nm demonstrated a power of 10 W.

Conversion of light into electricity in a semi-synthetic system based on photosynthetic bacterial chromatophores.

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 UQ0, disaccaride trehalose and antimycin A, an inhibitor of cytochrome bc1 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.

Protein-Mediated Carotenoid Delivery Suppresses the Photoinducible Oxidation of Lipofuscin in Retinal Pigment Epithelial Cells.

Lipofuscin of retinal pigment epithelium (RPE) cells is a complex heterogeneous system of chromophores which accumulates as granules during the cell's lifespan. Lipofuscin serves as a source of various cytotoxic effects linked with oxidative stress. Several age-related eye diseases such as macular degeneration of the retina, as well as some severe inherited eye pathologies, are accompanied by a significant increase in lipofuscin granule concentration. The accumulation of carotenoids in the RPE could provide an effective antioxidant protection against lipofuscin cytotoxic manifestations. Given the highly lipophilic nature of carotenoids, their targeted delivery to the vulnerable tissues can potentially be assisted by special proteins. In this study, we demonstrate how protein-mediated delivery of zeaxanthin using water-soluble Bombyx mori carotenoid-binding protein (BmCBP-ZEA) suppresses the photoinducible oxidative stress in RPE cells caused by irradiation of lipofuscin with intense white light. We implemented fluorescence lifetime imaging of the RPE cell culture ARPE-19 fed with lipofuscin granules and then irradiated by white light with and without the addition of BmCBP-ZEA. We demonstrate that after irradiation the mean fluorescence lifetime of lipofuscin significantly increases, while the presence of BmCBP-ZEA at 200 nM concentration suppresses the increase in the average lifetime of lipofuscin fluorescence, indicating an approx. 35% inhibition of the oxidative stress. This phenomenon serves as indirect yet important evidence of the efficiency of the protein-mediated carotenoid delivery into pigment epithelium cells.

Probing Red Blood Cell Membrane Microviscosity Using Fluorescence Anisotropy Decay Curves of the Lipophilic Dye PKH26.

Red blood cell (RBC) aggregation and deformation are governed by the molecular processes occurring on the membrane. Since several social important diseases are accompanied by alterations in RBC aggregation and deformability, it is important to develop a diagnostic parameter of RBC membrane structural integrity and stability. In this work, we propose membrane microviscosity assessed by time-resolved fluorescence anisotropy of the lipophilic PKH26 fluorescent probe as a diagnostic parameter. We measured the fluorescence decay curves of the PKH26 probe in the RBC membrane to establish the optimal parameters of the developed fluorescence assay. We observed a complex biphasic profile of the fluorescence anisotropy decay characterized by two correlation times corresponding to the rotational diffusion of free PKH26, and membrane-bounded molecules of the probe. The developed assay allowed us to estimate membrane microviscosity ηm in the range of 100-500 cP depending on the temperature, which paves the way for assessing RBC membrane properties in clinical applications as predictors of blood microrheological abnormalities.

Changes in the Electron Transfer Symmetry in the Photosystem I Reaction Centers upon Removal of Iron-Sulfur Clusters.

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 P700+ and phylloquinone acceptors A1A- and A1B- 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, FX, FA, and FB, and the PsaC protein subunit (the so-called P700-A1 core), in which phylloquinone molecules A1A and A1B serve as the terminal electron acceptors. It was shown that in the P700-A1 core preparations, the average distance between the centers of the P700+A1- 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 FX, FA, and FB are discussed.

Modulation of Membrane Microviscosity by Protein-Mediated Carotenoid Delivery as Revealed by Time-Resolved Fluorescence Anisotropy.

Carotenoids are potent antioxidants with a wide range of biomedical applications. However, their delivery into human cells is challenging and relatively inefficient. While the use of natural water-soluble carotenoproteins capable to reversibly bind carotenoids and transfer them into membranes is promising, the quantitative estimation of the delivery remains unclear. In the present work, we studied echinenone (ECN) delivery by cyanobacterial carotenoprotein AnaCTDH (C-terminal domain homolog of the Orange Carotenoid Protein from Anabaena), into liposome membranes labelled with BODIPY fluorescent probe. We observed that addition of AnaCTDH-ECN to liposomes led to the significant changes in the fast-kinetic component of the fluorescence decay curve, pointing on the dipole-dipole interactions between the probe and ECN within the membrane. It may serve as an indirect evidence of ECN delivery into membrane. To study the delivery in detail, we carried out molecular dynamics modeling of the localization of ECN within the lipid bilayer and calculate its orientation factor. Next, we exploited FRET to assess concentration of ECN delivered by AnaCTDH. Finally, we used time-resolved fluorescence anisotropy to assess changes in microviscosity of liposomal membranes. Incorporation of liposomes with ß-carotene increased membrane microviscosity while the effect of astaxanthin and its mono- and diester forms was less pronounced. At temperatures below 30 °C addition of AnaCTDH-ECN increased membrane microviscosity in a concentration-dependent manner, supporting the protein-mediated carotenoid delivery mechanism. Combining all data, we propose FRET-based analysis and assessment of membrane microviscosity as potent approaches to characterize the efficiency of carotenoids delivery into membranes.

In memory of Vladimir Anatolievich Shuvalov (1943-2022): an outstanding biophysicist.

We present here a tribute to one of the foremost biophysicists of our time, Vladimir Anatolievich Shuvalov, who made important contributions in bioenergetics, especially on the primary steps of conversion of light energy into charge-separated states in both anoxygenic and oxygenic photosynthesis. For this, he and his research team exploited pico- and femtosecond transient absorption spectroscopy, photodichroism & circular dichroism spectroscopy, light-induced FTIR (Fourier-transform infrared) spectroscopy, and hole-burning spectroscopy. We remember him for his outstanding leadership and for being a wonderful mentor to many scientists in this area. Reminiscences by many [Suleyman Allakhverdiev (Russia); Robert Blankenship (USA); Richard Cogdell (UK); Arvi Freiberg (Estonia); Govindjee Govindjee (USA); Alexander Krasnovsky, jr, (Russia); William Parson (USA); Andrei Razjivin (Russia); Jian- Ren Shen (Japan); Sergei Shuvalov (Russia); Lyudmilla Vasilieva (Russia); and Andrei Yakovlev (Russia)] have included not only his wonderful personal character, but his outstanding scientific research.

Current state of the primary charge separation mechanism in photosystem I of cyanobacteria.

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 Chl1A/Chl1B (dimer P700, or PAPB), Chl2A/Chl2B, and Chl3A/Chl3B 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 P700 is electronically coupled to the symmetrically located monomers Chl2A and Chl2B, which can be considered together as a symmetric exciplex Chl2APAPBChl2B with the mixed excited (Chl2APAPBChl2B)* and two charge-transfer states P700 +Chl2A - and P700 +Chl2B -. The redistribution of electrons between the branches in favor of the A-branch occurs after reduction of the Chl2A and Chl2B monomers. The formation of charge-transfer states and the symmetry breaking mechanisms were clarified by measuring the electrochromic Stark shift of ß-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.

Measurements of the light-induced steady state electric potential generation by photosynthetic pigment-protein complexes.

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.

Proton therapy with a fixed beamline for skull-base chordomas and chondrosarcomas: outcomes and toxicity.

AIM: This study presents an analysis (efficacy and toxicity) of outcomes in patients with skull-base chordomas or chondrosarcomas treated with a fixed horizontal pencil proton beam. BACKGROUND: Chordomas (CAs) and chondrosarcomas (CSAs) are rare tumours that are usually located near the base of the skull and very close to the brain's most critical structures. Proton therapy (PT) is often considered the best radiation treatment for these diseases, but it is still a limited resource. Active scanning PT delivered via a fixed pencil beamline might be a promising option. METHODS: This is a single-centre experience describing the results of proton therapy for 31 patients with CA (n = 23) or CSA (n = 8) located near the base of the skull. Proton therapy was utilized by a fixed pencil beamline with a chair to position the patient between May 2016 and November 2020. Ten patients underwent resection (32.2%), 15 patients (48.4%) underwent R2 resection, and 6 patients had unresectable tumours (19.4%). In 4 cases, the tumours had been previously irradiated. The median PT dose was 70 GyRBE (relative biological efficacy, 1.1) [range, 60 to 74] with 2.0 GyRBE per fraction. The mean GTV volume was 25.6 cm3 [range, 4.2-115.6]. Patient demographics, pathology, treatment parameters, and toxicity were collected and analysed. Radiation-induced reactions were assessed according to the Common Terminology Criteria for Adverse Events (CTCAE) v 4.0. RESULTS: The median follow-up time was 21 months [range, 4 to 52]. The median overall survival (OS) was 40 months. The 1- and 2-year OS was 100%, and the 3-year OS was 66.3%. Four patients died due to non-cancer-related reasons, 1 patient died due to tumour progression, and 1 patient died due to treatment-related injuries. The 1-year local control (LC) rate was 100%, the 2-year LC rate was 93.7%, and the 3-year LC rate was 85.3%. Two patients with CSA exhibited progression in the neck lymph nodes and lungs. All patients tolerated PT well without any treatment interruptions. We observed 2 cases of ≥ grade 3 toxicity, with 1 case of grade 3 myelitis and 1 case of grade 5 brainstem injury. CONCLUSION: Treatment with a fixed proton beam shows promising disease control and an acceptable toxicity rate, even the difficult-to-treat subpopulation of patients with skull-base chordomas or chondrosarcomas requiring dose escalation.

Generation of Photoelectric Responses by Photosystem II Core Complexes in the Presence of Externally Added Cytochrome c.

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 YZ to the primary quinone acceptor QA in the presence of oxidized cyt c (cyt c3+) 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 QA- to cyt c3+ 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 c2+ 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 c2+ to photooxidized tyrosine YZ. These data imply that cyt c3+ serves as a very effective exogenous electron acceptor for QA- in the case of intact PSII core complexes, and cyt c2+ is an extremely efficient artificial electron donor for YZ in the Mn-depleted PSII. The obtained data on the roles of cyt c2+ and cyt c3+ 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.

Phylloquinone is the principal Mehler reaction site within photosystem I in high light.

Photosynthesis is a vital process, responsible for fixing carbon dioxide, and producing most of the organic matter on the planet. However, photosynthesis has some inherent limitations in utilizing solar energy, and a part of the energy absorbed is lost in the reduction of O2 to produce the superoxide radical (O2•-) via the Mehler reaction, which occurs principally within photosystem I (PSI). For decades, O2 reduction within PSI was assumed to take place solely in the distal iron-sulfur clusters rather than within the two asymmetrical cofactor branches. Here, we demonstrate that under high irradiance, O2 photoreduction by PSI primarily takes place at the phylloquinone of one of the branches (the A-branch). This conclusion derives from the light dependency of the O2 photoreduction rate constant in fully mature wild-type PSI from Chlamydomonas reinhardtii, complexes lacking iron-sulfur clusters, and a mutant PSI, in which phyllosemiquinone at the A-branch has a significantly longer lifetime. We suggest that the Mehler reaction at the phylloquinone site serves as a release valve under conditions where both the iron-sulfur clusters of PSI and the mobile ferredoxin pool are highly reduced.

Symmetry breaking in photosystem I: ultrafast optical studies of variants near the accessory chlorophylls in the A- and B-branches of electron transfer cofactors.

Femtosecond absorption spectroscopy of Photosystem I (PS I) complexes from the cyanobacterium Synechocystis sp. PCC 6803 was carried out on three pairs of complementary amino acid substitutions located near the second pair of chlorophyll molecules Chl2A and Chl2B (also termed A-1A and A-1B). The absorption dynamics at delays of 0.1-500 ps were analyzed by decomposition into discrete decay-associated spectra and continuously distributed exponential components. The multi-exponential deconvolution of the absorption changes revealed that the electron transfer reactions in the PsaA-N600M, PsaA-N600H, and PsaA-N600L variants near the B-branch of cofactors are similar to those of the wild type, while the PsaB-N582M, PsaB-N582H, and PsaB-N582L variants near the A-branch of cofactors cause significant alterations of the photochemical processes, making them heterogeneous and poorly described by a discrete exponential kinetic model. A redistribution of the unpaired electron between the second and the third monomers Chl2A/Chl2B and Chl3A/Chl3B was identified in the time range of 9-20 ps, and the subsequent reduction of A1 was identified in the time range of 24-70 ps. In the PsaA-N600L and PsaB-N582H/L variants, the reduction of A1 occurred with a decreased quantum yield of charge separation. The decreased quantum yield correlates with a slowing of the phylloquinone A0 → A1 reduction, but not with the initial transient spectra measured at the shortest time delay. The results support a branch competition model, where the electron is sheared between Chl2A-Chl3A and Chl2B-Chl3B cofactors before its transfer to phylloquinone in either A1A or A1B sites.