Exciton interactions of chlorophyll tetramer in water-soluble chlorophyll-binding protein BoWSCP.

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

The donor-acceptor dyad based on high substituted fullero[70]pyrrolidine-coordinated manganese (III) phthalocyanine for photoinduced electron transfer.

Donor-acceptor dyads based on manganese porphyrins/phthalocyanines and fullerene derivatives with N-basicity centers have proved as promising photoinduced electron-transfer systems for photovoltaic devices, biologically active compounds, and molecular magnetic materials. The macroheterocyclic chromophore characterized by rich UV-visible-near IR absorption is the basis for the applications above. The problem of the synthesis and the characterization of new effective dyads was solved in this work on the example of the self-organizing system consisting of (octakis-3,5-di-tert-butylphenoxy)phthalocyaninato)manganese(III) acetate, (AcO)MnPc(3,5-di-tBuPhO)8, 2',5-di(pyridin-2'-yl)-3,4-fullero[70]pyrrolidine, Py2C70, and toluene. The phthalocyanine-fullerene dyads in the molecular and cationic form (respectively (AcO)(Py2C70)MnPc(3,5-di-tBuPhO)8 and [(Py2C70)MnPc(3,5-di-tBuPhO)8]+(AcO)-) were observed and described using the chemical kinetics/thermodynamics, UV-vis, IR, 1H NMR spectroscopy and mass spectrometry methods. The 1: 1 stoichiometry of both dyads was confirmed; the equilibrium and rate constant value, K= (4.86 ±â€¯0.56) × 104 L mol-1 and k = (4.455 ±â€¯3.37) × 10-5 s-1 was observed for the formation of molecular and cationic dyad, respectively. The study of (AcO)MnPc(3,5-di-tBuPhO)8 and [(Py2C70)MnPc(3,5-di-tBuPhO)8]+AcO- femtosecond transient absorption spectra points to the photoinduced electron transfer in the dyad, for which the lifetimes and the rate constants of charge separation (τCS, kCS) and charge recombination (τCR, kCR) were defined. The analysis of the relationship of the dyad physicochemical parameters with the molecular structure is represented using previously published data.

Femtosecond and Picosecond Dynamics of Recombinant Bacteriorhodopsin Primary Reactions Compared to the Native Protein in Trimeric and Monomeric Forms.

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

[Production of Gold Nanoparticles by Biogenesis Using Bacteria].

Diazotrophic cyanobacteria Anabaena sp. PCC 7120, four Nostoc strains, and two Azotobacter species (A. vinelandii and A. chroococcum) were found to produce gold nanoparticles (GNP) under nitrogen fixation conditions. GNP biogenesis occurred at AuHCl4 concentrations from 0.1 to 1 mM. In the cultures of unicellular cyanobacteria Synechococcus sp. PCC 7942 and Synechocystis incapable of nitrogen fixation, no GNP were formed at the same concentrations of gold salts. The plasmon resonance band peak was located at 552 nm. This position is characteristic of spherical GNP 10 to 30 nm in size. Small amounts of GNP were also formed in the culture liquid supernatants of the tested nitrogen-fixing bacteria at AuHCl4concentrations from 0.25 to 0.5 mM.

Impact of tightly focused femtosecond laser pulses on nucleolus-like bodies of mouse GV oocyte and the ability of mouse oocytes to mature.

Using femtosecond laser radiation, nucleolus-like bodies (NLBs) of mouse oocytes were locally dissected without damage to zona pellucida, cytoplasmic membrane, nuclear membrane, and nucleoplasm surrounding NLB. It was found that, after dissection of 2.7 × 10(-11) cm(3) of NLB material, which is approximately 5.2% of 10 µm NLB volume, the probability of germinal vesicle oocyte development to metaphase II stage of meiosis decreased 3-7 times compared to the non-treated oocytes. This result indicates that NLB material organization is significant for mouse oocyte maturation.

Quantum-classical model of retinal photoisomerization reaction in visual pigment rhodopsin.

A quantum-classical model of photoisomerization of the visual pigment rhodopsin chromophore is proposed. At certain (and more realistic) parameter value combinations, the model is shown to accurately reproduce a number of independent experimental data on the photoreaction dynamics: the quantum yield, the time to reach the point of conical intersection of potential energy surfaces, the termination time of the evolution of quantum subsystem, as well as the characteristic low frequencies of retinal molecular lattice fluctuations during photoisomerization. In addition, the model behavior is in good accordance with experimental data about coherence and local character of quantum transition.

[STIMULATION OF HaCaT KERATINOCYTES AND RATS MESENCHYMAL STROMAL CELLS PROLIFERATION BY FEMTOSECOND LASER PULSES].

The influence of femtosecond laser pulses on the proliferative activity of HaCaT keratinocytes and mesenchymal stromal cells (MSC) rats was studied. The growth media was exposed by laser pulses with wavelength 590 nm and duration 30 fs. The dependence of proliferative activity of cells on the dose was showed in the range 6-4299 J/cm2. Proliferative activity was assessed by the number of cells after 1 day after exposure. For both cell cultures obtained similar dose dependence: an increase in cell proliferation (32-54% for HaCaT and 19% for MSK) occurs when using lower doses, while higher doses no changes the rate of proliferation of cells. Conducted physical and chemical analysis found no increase in the concentration of active forms of oxygen in the culture medium. The impact of femtosecond laser pulses has led to the generation in culture medium acoustic oscillations in the range of 0.5 to 6.0 kHz. It is assumed that the increase in proliferative activity of cells, can be caused by mechanical effects of acoustic waves generated in the environment of optical breakdown in the focus of the laser radiation.

Electron transfer in photosystem I containing native and modified quinone acceptors.

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.

Effect of laser optoperforation of the zona pellucida on mouse embryo development in vitro.

The effect of laser optical perforation of the zona pellucida on the viability and development of mouse embryos has been studied. Operations of zona pellucida thinning and single or double perforation were carried out on 2-cell embryo, morula, and blastocyst stages with a laser pulse (wavelength 1.48 µm, pulse duration 2 ms). Embryo development up to the blastocyst stage and hatching efficiency were statistically analyzed. It was found that 2-cell or morula stage embryo zona pellucida thinning or single perforation did not affect development to the blastocyst stage and number of hatched embryos, but it accelerated embryo hatching compared to control groups one day earlier in vitro. Double optoperforation on 2-cell embryo or morula stage did not significantly affect development to the blastocyst stage, but it strongly decreased the number of hatched embryos. Also, zona pellucida perforation at the blastocyst stage had a negative effect: hatching did not occur after this manipulation. Blastocyst cell number calculation after single zona pellucida perforation at 2-cell and morula stages showed that cell number of hatching or hatched blastocysts did not differ from the same control groups. This fact points out that the laser single optoperforation method is a useful and safe experimental tool that allows further manipulations within the zona pellucida.

Primary radical ion pairs in photosystem II core complexes.

Ultrafast absorption spectroscopy with 20-fs resolution was applied to study primary charge separation in spinach photosystem II (PSII) reaction center (RC) and PSII core complex (RC complex with integral antenna) upon excitation at maximum wavelength 700-710 nm at 278 K. It was found that the initial charge separation between P680* and ChlD1 (Chl-670) takes place with a time constant of ~1 ps with the formation of the primary charge-separated state P680* with an admixture of: P680*((1-δ)) (P680(δ+)ChlD1(δ-)), where δ ~ 0.5. The subsequent electron transfer from P680(δ+)ChlD1(δ-) to pheophytin (Pheo) occurs within 13 ps and is accompanied by a relaxation of the absorption band at 670 nm (ChlD1(δ-)) and bleaching of the PheoD1 bands at 420, 545, and 680 nm with development of the Pheo(-) band at 460 nm. Further electron transfer to QA occurs within 250 ps in accordance with earlier data. The spectra of P680(+) and Pheo(-) formation include a bleaching band at 670 nm; this indicates that Chl-670 is an intermediate between P680 and Pheo. Stimulated emission kinetics at 685 nm demonstrate the existence of two decaying components with time constants of ~1 and ~13 ps due to the formation of P680(δ+)ChlD1(δ-) and P680(+)PheoD1(-), respectively.

Formation and decay of P680 (P(D1)-P(D2))⁺PheoD1⁻ radical ion pair in photosystem II core complexes.

Under physiological conditions (278 K) femtosecond pump-probe laser spectroscopy with 20-fs time resolution was applied to study primary charge separation in spinach photosystem II (PSII) core complexes excited at 710 nm. It was shown that initial formation of anion radical band of pheophytin molecule (Pheo⁻) at 460 nm is observed with rise time of ~11ps. The kinetics of the observed rise was ascribed to charge separation between Chl (chlorophyll a) dimer, primary electron donor in PSII (P680*) and Pheo located in D1 protein subunit (PheoD1) absorbing at 420 nm, 545 nm and 680 nm with formation of the ion-radical pair P680⁺PheoDI⁻. The subsequent electron transfer from Pheo(D1)⁻ to primary plastoquinone electron acceptor (Q(A)) was accompanied by relaxation of the 460-nm band and occurred within ~250 ps in good agreement with previous measurements in Photosystem II-enriched particles and bacterial reaction centers. The subtraction of the P680⁺ spectrum measured at 455 ps delay from the spectra at 23 ps or 44 ps delay reveals the spectrum of Pheo(DI)⁻, which is very similar to that measured earlier by accumulation method. The spectrum of Pheo(DI)⁻ formation includes a bleaching (or red shift) of the 670 nm band indicating that Chl-670 is close to Pheo(D1). According to previous measurements in the femtosecond-picosecond time range this Chl-670 was ascribed to Chl(D1) [Shelaev, Gostev, Vishnev, Shkuropatov, Ptushenko, Mamedov, Sarkisov, Nadtochenko, Semenov and Shuvalov, J. Photochemistry and Photobiology, B: Biology 104 (2011) 45-50]. Stimulated emission at 685 nm was found to have two decaying components with time constants of ~1ps and ~14ps. These components appear to reflect formation of P680⁺Chl(D1)⁻ and P680⁺Pheo(D1)⁻, respectively, as found earlier. This article is part of a special issue entitled: photosynthesis research for sustainability: keys to produce clean energy.

Antibacterial effects of silver nanoparticles on gram-negative bacteria: influence on the growth and biofilms formation, mechanisms of action.

Antibacterial action of silver nanoparticles (AgNP) on Gram-negative bacteria (planctonic cells and biofilms) is reported in this study. AgNP of 8.3 nm in diameter stabilized by hydrolyzed casein peptides strongly inhibited biofilms formation of Escherichia coli AB1157, Pseudomonas aeruginosa PAO1 and Serratia proteamaculans 94 in concentrations of 4-5 µg/ml, 10 µg/ml and 10-20 µg/ml, respectively. The viability of E. coli AB1157 cells in biofilms was considerably reduced by AgNP concentrations above 100 to -150 µg/ml. E. coli strains with mutations in genes responsible for the repair of DNA containing oxidative lesions (mutY, mutS, mutM, mutT, nth) were less resistant to AgNP than wild type strains. This suggests that these genes may be involved in the repair of DNA damage caused by AgNP. E. coli mutants deficient in excision repair, SOS-response and in the synthesis of global regulators RpoS, CRP protein and Lon protease present similar resistance to AgNP as wild type cells. LuxI/LuxR Quorum Sensing systems did not participate in the control of sensitivity to AgNP of Pseudomonas and Serratia. E. coli mutant strains deficient in OmpF or OmpC porins were 4-8 times more resistant to AgNP as compared to the wild type strain. This suggests that porins have an important function related AgNP antibacterial effects.

[Obtainment of chimeric blastocysts of mice by methods of laser nanosurgery].

The procedure of obtainment of chimeric blastocysts of mice by laser nanosurgery methods without using any other techniques is described. To perform the experiments, a special laser micromanipulator was invented. The murine embryonic stem cells (ESC), which were transformed with pEF-GFP vector, encoding the green fluorescent protein, were used in the experiments. ESC were introduced into the perivitelline space of murine embryos at the stage of 8 cells using the laser micromanipulator. The operated embryos were cultured in vitro until the stage of emergence from zona pellucida. The fluorescence and its precise localization were registered using a confocal microscope. It was shown for the first time that the inclusions of ESC introduced with the lased micromanipulator were found not only in the inner cell mass (ICM) but also in the trophectoderm of the chimeric blastocyst. The technology of nanosurgical operations at early stage preimplanted mammalian embryos using laser techniques opens great opportunities not only for solution of fundamental tasks of experimental embryology of mammals but also for obtainment of chimeric and transgenic animals with predetermined genotype.

Primary steps of electron and energy transfer in photosystem I: effect of excitation pulse wavelength.

Time-resolved differential spectra of photosystem I complex were obtained by the "pump-probe" technique with 25-fs pulses with maxima at 670, 700, and 720 nm. The ratio between the number of excited chlorophyll molecules of the antenna and of the reaction center was shown to depend on spectral characteristics of the pump pulses. In all cases, an ultrafast (<150 fs) formation of the primary radical pair P700(+)A(0)() was recorded. However, on excitation by pulses with maxima at 670 or 700 nm, detection of the charge separation was masked by the much more intensive bleaching at the chlorophyll Q(y) band due to excitation of the bulk antenna chlorophylls. We show that triggering the charge separation by 25-fs pulses centered at 720 nm allows to detect more clearly kinetics of formation of the primary and secondary ion-radical pairs. The findings help to explain possible reasons for discrepancies of kinetics of primary steps of electron transfer detected in different laboratories.

P680 (P(D1)P(D2)) and Chl(D1) as alternative electron donors in photosystem II core complexes and isolated reaction centers.

Low temperature (77-90 K) measurements of absorption spectral changes induced by red light illumination in isolated photosystem II (PSII) reaction centers (RCs, D1/D2/Cyt b559 complex) with different external acceptors and in PSII core complexes have shown that two different electron donors can alternatively function in PSII: chlorophyll (Chl) dimer P(680) absorbing at 684 nm and Chl monomer Chl(D1) absorbing at 674 nm. Under physiological conditions (278 K) transient absorption difference spectroscopy with 20-fs resolution was applied to study primary charge separation in spinach PSII core complexes excited at 710 nm. It was shown that the initial electron transfer reaction takes place with a time constant of ~0.9 ps. This kinetics was ascribed to charge separation between P(680)* and Chl(D1) absorbing at 670 nm accompanied by the formation of the primary charge-separated state P(680)(+)Chl(DI)(-), as indicated by 0.9-ps transient bleaching at 670 nm. The subsequent electron transfer from Chl(D1)(-) occurred within 13-14 ps and was accompanied by relaxation of the 670-nm band, bleaching of the Pheo(D1) Q(x) absorption band at 545 nm, and development of the anion-radical band of Pheo(D1)(-) at 450-460 nm, the latter two attributable to formation of the secondary radical pair P(680)(+)Pheo(D1)(-). The 14-ps relaxation of the 670-nm band was previously assigned to the Chl(D1) absorption in isolated PSII RCs [Shelaev, Gostev, Nadtochenko, Shkuropatov, Zabelin, Mamedov, Semenov, Sarkisov and Shuvalov, Photosynth. Res. 98 (2008) 95-103]. We suggest that the longer wavelength position of P(680) (near 680 nm) as a primary electron donor and the shorter wavelength position of Chl(D1) (near 670 nm) as a primary acceptor within the Q(y) transitions in RC allow an effective competition with an energy transfer and stabilization of separated charges. Although an alternative mechanism of charge separation with Chl(D1)* as the primary electron donor and Pheo(D1) as the primary acceptor cannot be ruled out, the 20-fs excitation at the far-red tail of the PSII core complex absorption spectrum at 710 nm appears to induce a transition to a low-energy state P(680)* with charge-transfer character (probably P(D1)(δ+)P(D2)(δ-)) which results in an effective electron transfer from P(680)* (the primary electron donor) to Chl(D1) as the intermediary acceptor.

Femtosecond formation dynamics of primary photoproducts of visual pigment rhodopsin.

The coherent 11-cis-retinal photoisomerization dynamics in bovine rhodopsin was studied by femtosecond time-resolved laser absorption spectroscopy at 30-fs resolution. Femtosecond pulses of 500, 535, and 560 nm wavelength were used for rhodopsin excitation to produce different initial Franck-Condon states and relevant distinct values of the vibrational energy of the molecule in its electron excited state. Time evolution of the photoinduced rhodopsin absorption spectra was monitored after femtosecond excitation in the spectral range of 400-720 nm. Oscillations of the time-resolved absorption signals of rhodopsin photoproducts represented by photorhodopsin(570) with vibrationally-excited all-trans-retinal and rhodopsin(498) in its initial state with vibrationally-excited 11-cis-retinal were studied. These oscillations reflect the dynamics of coherent vibrational wave-packets in the ground state of photoproducts. Fourier analysis of these oscillatory components has revealed frequencies, amplitudes, and initial phases of different vibrational modes, along which the motion of wave-packets of both photoproducts occurs. The main vibrational modes established are 62, 160 cm(-1) and 44, 142 cm(-1) for photorhodopsin(570) and for rhodopsin(498), respectively. These vibrational modes are directly involved in the coherent reaction under the study, and their amplitudes in the power spectrum obtained through the Fourier transform of the kinetic curves depend on the excitation wavelength of rhodopsin.