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%.

Specificity of Photochemical Energy Conversion in Photosystem I from the Green Microalga Chlorella ohadii.

Primary excitation energy transfer and charge separation in photosystem I (PSI) from the extremophile desert green alga Chlorella ohadii grown in low light were studied using broadband femtosecond pump-probe spectroscopy in the spectral range from 400 to 850 nm and in the time range from 50 fs to 500 ps. Photochemical reactions were induced by the excitation into the blue and red edges of the chlorophyll Qy absorption band and compared with similar processes in PSI from the cyanobacterium Synechocystis sp. PCC 6803. When PSI from C. ohadii was excited at 660 nm, the processes of energy redistribution in the light-harvesting antenna complex were observed within a time interval of up to 25 ps, while formation of the stable radical ion pair P700+A1- was kinetically heterogeneous with characteristic times of 25 and 120 ps. When PSI was excited into the red edge of the Qy band at 715 nm, primary charge separation reactions occurred within the time range of 7 ps in half of the complexes. In the remaining complexes, formation of the radical ion pair P700+A1- was limited by the energy transfer and occurred with a characteristic time of 70 ps. Similar photochemical reactions in PSI from Synechocystis 6803 were significantly faster: upon excitation at 680 nm, formation of the primary radical ion pairs occurred with a time of 3 ps in ~30% complexes. Excitation at 720 nm resulted in kinetically unresolvable ultrafast primary charge separation in 50% complexes, and subsequent formation of P700+A1- was observed within 25 ps. The photodynamics of PSI from C. ohadii was noticeably similar to the excitation energy transfer and charge separation in PSI from the microalga Chlamydomonas reinhardtii; however, the dynamics of energy transfer in C. ohadii PSI also included slower components.

Reversible Photochromic Reactions of Bacteriorhodopsin from Halobacterium salinarum at Femto- and Picosecond Times.

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

Femtosecond Dynamics of Excited States of Chlorophyll Tetramer in Water-Soluble Chlorophyll-Binding Protein BoWSCP.

The paper reports on the absorption dynamics of chlorophyll a in a symmetric tetrameric complex of the water-soluble chlorophyll-binding protein BoWSCP. It was measured by a broadband femtosecond laser pump-probe spectroscopy within the range from 400 to 750 nm and with a time resolution of 20 fs-200 ps. When BoWSCP was excited in the region of the Soret band at a wavelength of 430 nm, nonradiative intramolecular conversion S3→S1 was observed with a characteristic time of 83 ± 9 fs. When the complex was excited in the region of the Qy band at 670 nm, relaxation transition between two excitonic states of the chlorophyll dimer was observed in the range of 105 ± 10 fs. Absorption spectra of the excited singlet states S1 and S3 of chlorophyll a were obtained. The delocalization of the excited state between exciton-coupled Chl molecules in BoWSCP tetramer changed in time and depended on the excitation energy. When BoWSCP is excited in the Soret band region, an ultrafast photochemical reaction is observed. This could result from the reduction of tryptophan in the vicinity of chlorophyll.

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.

Comparative Absorption Dynamics of the Singlet Excited States of Chlorophylls a and d.

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 S1 singlet states of chlorophylls a and d were recorded, and the dynamics of the of the Qy 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 Qx→Qy (electronic transition S2→S1) was measured; the characteristic relaxation time was 54 ± 3 and 45 ± 9 fs for chlorophylls a and d, respectively.

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.

Intramolecular photo-driven charge transfer in a series of pyridyl substituted phenyloxazoles. Structural relaxation in meta-substituted ethylpyridinium derivative of phenyloxazole.

A series of pyridyl (pyridinium) substituted benzoxazoles were studied by steady state absorption, fluorescence spectroscopy, time-resolved fluorescence spectroscopy, fs pulse absorption and polarization spectroscopy, and quantum-chemical calculations. The spectral and kinetic parameters of the fluorophores in MeCN and EtOAc were obtained experimentally and were calculated by means of DFT and TDDFT methods. A scheme including four transient excited states was proposed for the interpretation of differential absorption kinetics of the charged fluorophores. Expressions describing the actual kinetics graphs, the decay associated spectra, and the species-associated spectra were derived. The charge shift step was found to be dependent on average solvation times. A charge shift followed by the formation of the twisted conformer was found for the excited 1-ethyl-3-(5-phenyloxazol-2-yl)pyridinium 4-methyl-1-benzenesulfonate in MeCN and EtOAc. Conformational analysis confirms a large amplitude motion of the meta-substituted ethylpyridinium group as an additional structural relaxation path producing an abnormally large fluorescence Stokes shift.

Unprecedented Coordination-Induced Bright Red Emission from Group 12 Metal-Bound Triarylazoimidazoles.

Arylazoimidazoles are important dyes which were intensively studied in the past. In contrast, triarylazoimidazoles (derivatives which carry aryl substituents at the imidazole core) received almost no attention in the scientific literature. Here, we report a new family of simple and easily accessible triarylazoimidazole-group 12 metal complexes, which feature highly efficient photo-luminescence emission (Φ up to 0.44). Novel compounds exhibit bright red emission in solution, which could be excited with a visible light.

PSI-SMALP, a Detergent-free Cyanobacterial Photosystem I, Reveals Faster Femtosecond Photochemistry.

Cyanobacterial photosystem I (PSI) functions as a light-driven cyt c6-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-ß-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 P700. 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 P700 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.

Generation of ion-radical chlorophyll states in the light-harvesting antenna and the reaction center of cyanobacterial photosystem I.

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 ß-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 P700+A1- at 40 ps was limited by energy transfer from LWC to the primary donor P700 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 PB+Chl2B-, where negative charge is localized predominantly at the accessory chlorophyll molecule in the branch B, Chl2B. The following decrease of carotenoid signal at ~ 5 ps was ascribed to electron transfer to the more distant molecule Chl3B. The reduction of phylloquinone in the sites A1A and A1B 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 PB+A1A-.

Femtosecond excited state dynamics of stilbene-viologen complexes with a weakly pronounced charge transfer.

The femtosecond dynamics of photoinduced electron transfers in supramolecular donor-acceptor complexes between (E)-bis(18-crown-6)stilbene (D) and tetraperchlorates of 2,7-di(2-ammonioethyl)(2,7-diazapyrenium) (A1), 3,3'-(E)-ethene-1,2-diylbis[1-(3-ammoniopropyl)pyridinium] (A2) and 4,4'-ethane-1,2-diylbis[1-(3-ammoniopropyl)pyridinium] (A3) was studied. The acceptors A2 and A3 are weak electron acceptors whose first reduction potentials are equal to -1.0 and -1.2 V (Ag), respectively, while A1 is a strong acceptor with a reduction potential of -0.42 V. It was shown that the back electron transfer time in CT-states of the complexes D·A2 and D·A3 is 30-40 ps, which is approximately 50 times greater than the analogous time for the charge transfer complexes studied earlier. The complex D·A1 is characterized by ultrafast back electron transfer (770 fs). The relaxation pathway of excited states of D·A1 depends on the wavelength of the excitation light. When excited at 356 nm, the accumulation of a transient locally excited (LE) state with a 250 fs lifetime was observed. But when excited at 425 nm, the formation of the LE-state was not observed.

A novel approach for 3D reconstruction of mice full-grown oocytes by time-of-flight secondary ion mass spectrometry.

Currently two techniques exist for 3D reconstruction of biological samples by time-of-flight secondary ion mass spectrometry (ToF-SIMS). The first, based on microtomy and combining of successive section images, is successfully applied for tissues, while the second, based on sputter depth profiling, is widely used for cells. In the present work, we report the first successful adaptation of sectioning technique for ToF-SIMS 3D imaging of a single cell-fully grown mouse germinal vesicle (GV) oocyte. In addition, microtomy was combined with sputter depth profiling of individual flat sections for three-dimensional reconstruction of intracellular organelles. GV oocyte sectioning allowed us to obtain molecule-specific 3D maps free from artifacts associated with surface topography and uneven etching depth. Sputter depth profiling of individual flat slices revealed fine structure of specific organelles inside the oocyte. Different oocyte organelles (cytoplasm, germinal vesicle, membranes, cumulus cells) were presented on the ion images. Atypical nucleoli referred to as "nucleolus-like body" (NLB) was detected inside the germinal vesicle in PO3- and CN- ions generated by nucleic acids and proteins respectively. Significant difference in PO3- intensity in the NLB central area and NLB border was found. This difference appears as a bright halo around the center area. The NLB size calculated for PO3- and CN- ion images is 12.9 ± 0.2 µm and 11.9 ± 0.2 µm respectively, which suggests that bright halo of PO3- ions is a chromatin compaction on the NLB surface. Areas of approximately 1.0-2.5 µm size inside nucleoplasm with increased PO3- and CN- signal were registered in germinal vesicle. Observed compartments have different sizes and shapes, and they are likely attributed to chromocenters or chromosomes.

Mechanism of adiabatic primary electron transfer in photosystem I: Femtosecond spectroscopy upon excitation of reaction center in the far-red edge of the QY band.

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 P700+A0- at time delay <180fs, followed by formation of the secondary pair P700+A1- with a characteristic time of 26ps. The obtained data are rationalized in the framework of adiabatic three-state model that includes the chlorophyll dimer P700 and two symmetrically arranged nearest chlorophyll molecules of A0. The arrangement of chlorophylls results in strong electronic coupling between P700 and A0. Excitation in the maximum of P700 absorption generates electronic states with the highest contribution from P700*, whereas excitation in the far-red edge predominantly generates charge transfer state P700+A0- 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 P700 and A0, providing a microscopic explanation of the ultrafast formation of P700+A0- and exponential decline of PS I absorption.

Dynamics of excited-state intramolecular proton-transfer in 2-amino-3-(2'-benzazolyl)quinoline cations.

It was found that cations formed by the protonation of 2-amino-3-(2'-benzoxazolyl)-quinoline (ABO) and 2-amino-3-(2'-benzothiazolyl)-quinoline (ABT) at the nitrogen atom of the quinoline ring exhibit excited-state intramolecular proton transfer (ESIPT). The two-band fluorescence of these cations is due to the emission from two species: the initial tautomer (short-wavelength band) and the ESIPT product (long-wavelength band). The relative intensity of the long-wavelength band depends on the basicity of the proton-accepting moiety and temperature. Quantum-chemical calculations demonstrated that ESIPT in cations involves overcoming a significant potential barrier, which increases with the decreasing basicity of the proton-accepting benzazole moiety. Using femtosecond absorption spectroscopy and nanosecond fluorescence spectroscopy, the effective ESIPT time in the studied cations was determined, which increased with decreasing temperature.

Femtosecond excited state dynamics of a stilbene-viologen charge transfer complex assembled via host-guest interaction.

The dynamics of the excited states of a supramolecular complex with a charge transfer between (E)-bis(18-crown-6)stilbene and 4,4'-(E)-ethene-1,2-diylbis[1-(2-ammonioethyl)pyridinium]tetraperchlorate was studied by means of femtosecond transient spectroscopy. It is found that the characteristic time of the conversion of the locally excited (LE) state into the charge transfer (CT) state is equal to 300 fs, whereas the characteristic time of the conversion of the CT state into the ground state is equal to 400 fs. Due to host-guest interaction involving hydrogen bonds, the complex possesses high thermodynamic stability. As a result of ultrafast photoinduced processes of the direct and back electron transfer, the complex does not fluoresce. Upon the interaction of the complex with alkaline-earth metal cations, "switch-on" of its fluorescence occurs.

Correlating microscopy techniques and ToF-SIMS analysis of fully grown mammalian oocytes.

The 2D-molecular thin film analysis protocol for fully grown mice oocytes is described using an innovative approach. Time-of-flight secondary ion mass spectrometry (ToF-SIMS), scanning electron microscopy (SEM), atomic force microscopy (AFM) and optical microscopy imaging were applied to the same mice oocyte section on the same sample holder. A freeze-dried mice oocyte was infiltrated into embedding media, e.g. Epon, and then was cut with a microtome and 2 µm thick sections were transferred onto an ITO coated conductive glass. Mammalian oocytes can contain "nucleolus-like body" (NLB) units and ToF-SIMS analysis was used to investigate the NLB composition. The ion-spatial distribution in the cell components was identified and compared with the images acquired by SEM, AFM and optical microscopy. This study presents a significant advancement in cell embryology, cell physiology and cancer-cell biochemistry.

Time-of-flight secondary ion mass spectrometry to assess spatial distribution of A2E and its oxidized forms within lipofuscin granules isolated from human retinal pigment epithelium.

Lipofuscin granules accumulate in the cells of retinal pigment epithelium with age, particularly in patients with hereditary diseases. These granules are heterogeneous, being composed of mixtures of proteins and lipids, including more than 21 different fluorescent compounds. Bisretinoids and their photo-oxidation and photodegradation products represent the main source of lipofuscin fluorescence and exhibit phototoxic properties. This study used time-of-flight secondary ion mass spectrometry (ToF-SIMS) with in-depth probing to assess the depth distribution of N-retinylidene-N-retinylethanolamine (A2E) and its singly and doubly oxidized forms (A2E-ox and A2E-2ox, respectively) within lipofuscin granules and in their surface layer (lipid membrane). ToF-SIMS showed that A2E and its oxidized forms were uniformly distributed throughout lipofuscin granules but were not present at the membrane surface layer. This finding is important for understanding the process involved in the formation of lipofuscin granules and in their toxicity.

Excitonic Coupling and Femtosecond Relaxation of Zinc Porphyrin Oligomers Linked with Triazole Bridge: Dynamics and Modeling.

The synthesis of new zinc porphyrin oligomers linked by a triazole bridge was carried out via "click" reaction. A split in the porphyrin oligomer B-band was observed. It was considered as evidence of exciton-excitonic coupling. The relaxation of excited states in Q-band porphyrin oligomers was studied by the femtosecond laser spectroscopy technique with a 20 fs pump pulse. The transient oscillations of two B-band excitonic peaks have a π-radian shift. For explanation of the coherent oscillation, a theoretical model was developed. The model considered the combination of the exciton-excitonic coupling between porphyrin rings in dimer and weak exciton-vibronic coupling in one porphyrin ring. By varying the values of the structural parameters of porphyrins (the strength values of this couplings and measure of symmetry breaking), we obtained correspondence between the experimental data (phase shift and amplitudes of the spectrum oscillations) and the predictions of the model developed here.

Evidence that histidine forms a coordination bond to the A(0A) and A(0B) chlorophylls and a second H-bond to the A(1A) and A(1B) phylloquinones in M688H(PsaA) and M668H(PsaB) variants of Synechocystis sp. PCC 6803.

The axial ligands of the acceptor chlorophylls, A(0A) and A(0B), in Photosystem I are the Met sulfur atoms of M688(PsaA) and M668(PsaB). To determine the role of the Met, His variants were generated in Synechocystis sp. PCC 6803. Molecular dynamics simulations on M688H(PsaA) show that there exist low energy conformations with the His coordinated to A(0A) and possibly H-bonded to A(1A). Transient EPR studies on M688H(PsaA) indicate a more symmetrical electron spin distribution in the A(1A) phyllosemiquinone ring consistent with the presence of an H-bond to the C1 carbonyl. Ultrafast optical studies on the variants show that the 150fs charge separation between P700 and A(0) remains unaffected. Studies on the ns timescale show that 57% of the electrons are transferred from A(0A)(-) to A(1A) in M688H(PsaA) and 48% from A(0B)(-) to A(1B) in M668H(PsaB); the remainder recombine with P700(+) with 1/e times of 25ns and 37ns, respectively. Those electrons that reach A(1A) and A(1B) in the branch carrying the mutation are not transferred to FX, but recombine with P700(+) with 1/e times of ~15µs and ~5µs, respectively. Hence, the His is coordinated to A0 in all populations, but in a second population, the His may be additionally H-bonded to A(1). Electron transfer from A(0) to A(1) occurs only in the latter, but the higher redox potentials of A(0) and A(1) as a result of the stronger coordination bond to A(0) and the proposed second H-bond to A(1) preclude electron transfer to the Fe/S clusters.