A.B. Rubin

Evidence for a correlation between the photoinduced electron transfer and dynamic properties of the chromatophore membranes from Rhodospirillum rubrum.

Besides the study of the static structure of biologically important macromolecules, investigation of the dynamic properties has gained much interest in the last few years. X-ray diffraction on myo~obin crystals [l] revealed structural dynamics. Evidence of a large number of confo~ational substates of a molecule was found. Molecules in different conformational substates have the same gross structure but differ in local configurations. To separate the static from the dynamic disorder in the myoglobin crystals, X-ray data and Mossbauer measurements on the heme iron of myoglobin were compared [ 11. It was shown that these Mossbauer experiments themselves gave valuable information on the dynamic properties of the system [2]. The iron labels the motions of the molecule in a specific im~rtant place. In contrast to the study of the dynamic properties on a well-known molecule like myoglobin, the investigations on membrane-bound proteins are much more complicated. Many systems are not described completely even with respect to their biochemical properties. Energy accumulation by photosynthesis demonstrates the importance of these molecules in nature. A large number of membrane-bound proteins involved in the electron transport after light absorption contain iron. The Mijssbauer spectroscopy is, therefore, a valuable tool in the study of such systems, especially with respect to dynamic properties. X-ray ~vestigations, as in [l], cannot be applied because the whole system cannot be crystallized. Despite the large complexity of such systems an interpretation of

Membrane lipid peroxidation, cell viability and Photosystem II activity in the green alga Chlorella pyrenoidosa subjected to various stress conditions

Abstract The unicellular green alga Chlorella pyrenoidosa was subjected to a variety of stress conditions (strong illumination, incubation with Cu 1+ or Zn 2+ , exposure to high temperatures). The amplitude of thermoluminescence (TL) peak at 125°C, accumulation of thiobarbituric acid reactive substances (TBARS), which indicate an accumulation of lipid peroxidation products, efficiency of Photosystem II reactions ( F v / F M ratio) and the percentage of viable cells were measured in stressed culture. Exposure of algae to strong (5000 μmol photons m 2 s 1 ) or to low (60 μmol photons m −2 s −1 ) light combined with the addition of 1.6 μM Cu 2+ or 30 μM Zn 2+ inactivated Photosystem II, decreased the viability of Chlorella cells, and, finally, significantly enhanced TL and the accumulation of TBARS, which was accompanied by chlorophyll bleaching. TL emission started to rise after a lag-period of about 30 min in algae subjected to strong illumination, 2–3 h in copper-treated algae, and 10 h in zinc-treated algae. A vast majority of cells were nonviable to the end of the lag-period. The addition of Cu 2+ or ZN 2+ in darkness caused a slight decrase in the F v / F M ratio without significant changes in TL emission. Incubation of algae at 50°C for 10 min did not affect the F v / F M ratio nd cell viability, whereas no viable cells and Photosystem II activity were detected in the culture incubated at 55°C. Heat stress at temperatures above 55°C significantly enhanced the amplitude of the 125°C TL peak and the accumulation of TBARS when the algae were further incubated at low light at room temperature. We conclude that, under the stress conditions used in this study, (i) lipid peroxides and products of their degradation are not responsible for the cytolethal effect in Chlorella and (ii) lipid peroxidation arises mainly upon illumination of dead cells.

Targeting Cancer Cells by Novel Engineered Modular Transporters

A major problem in the treatment of cancer is the specific targeting of drugs to these abnormal cells. Ideally, such a drug should act over short distances to minimize damage to healthy cells and target subcellular compartments that have the highest sensitivity to the drug. We describe the novel approach of using modular recombinant transporters to target photosensitizers to the nucleus, where their action is most pronounced, of cancer cells overexpressing ErbB1 receptors. We have produced a new generation of the transporters consisting of (a) epidermal growth factor as the internalizable ligand module to ErbB1 receptors, (b) the optimized nuclear localization sequence of SV40 large T-antigen, (c) a translocation domain of diphtheria toxin as an endosomolytic module, and (d) the Escherichia coli hemoglobin-like protein HMP as a carrier module. The modules retained their functions within the transporter chimera: they showed high-affinity interactions with ErbB1 receptors and alpha/beta-importin dimers and formed holes in lipid bilayers at endosomal pH. A photosensitizer conjugated with the transporter produced singlet oxygen and (*)OH radicals similar to the free photosensitizer. Photosensitizers-transporter conjugates have >3,000 times greater efficacy than free photosensitizers for target cells and were not photocytotoxic at these concentrations for cells expressing a few ErbB1 receptors per cell, in contrast to free photosensitizers. The different modules of the transporters, which are highly expressed and easily purified to retain full activity of each of the modules, are interchangeable, meaning that they can be tailored for particular applications.

Probing the kinetics of Photosystem I and Photosystem II fluorescence in pea chloroplasts on a picosecond pulse fluorometer

Picosecond fluorescence kinetics of pea chloroplasts have been investigated at room temperature using a pulse fluorometer with a resolution time of 10-11 s. Fluorescence has been excited by both a ruby and neodymium-glass mode-locked laser and has been reocrded within the 650 to 800 nm spectral region. We have found three-component kinetics of fluorescence from pea chloroplasts with lifetimes of 80, 300 and 4500 ps, respectively. The observed time dependency of the fluorescence of different components on the functional state of the photosynthetic mechanism as well as their spectra enabled us to conclude that Photosystem I fluoresces with a lifetime of 80 ps (tauI) and Photosystem II fluoresces with a lifetime of 300 ps (tauII). Fluorescence with a lifetime of 4500 ps (tauIII) may be interpreted as originating from chlorophill monomeric forms which are not involved in photosynthesis. It was determined that the rise time of Photosystem I and Photosystem II fluorescence after 530 nm photoexcitation is 200 ps, which corrsponds to the time of energy migration to them from carotenoids.

Hydrogen production by photoautotrophic sulfur‐deprived Chlamydomonas reinhardtii pre‐grown and incubated under high light

We have previously demonstrated that Chlamydomonas reinhardtii can produce hydrogen under strictly photoautotrophic conditions during sulfur deprivation [Tsygankov et al. (2006); Int J Hydrogen Energy 3:1574–1584]. The maximum hydrogen photoproduction was achieved by photoautotrophic cultures pre‐grown under a low light regime (25 µE m−2 s−1). We failed to establish sustained hydrogen production from cultures pre‐grown under high light (100 µE m−2 s−1). A new approach for sustained hydrogen production by these cultures is presented here. Assuming that stable and reproducible transition to anerobiosis as well as high starch accumulation are important for hydrogen production, the influence of light intensity and dissolved oxygen concentration during the oxygen evolving stage of sulfur deprivation were investigated in cultures pre‐grown under high light. Results showed that light higher than 175 µE m−2 s−1 during sulfur deprivation induced reproducible transition to anerobiosis, although the total amount of starch accumulation and hydrogen production were insignificant. The potential PSII activity measured in the presence of an artificial electron acceptor (DCBQ) and an inhibitor of electron transport (DBMIB) did not change in cultures pre‐grown under 20 µE m−2 s−1 and incubated under 150 µE m−2 s−1 during sulfur deprivation. In contrast, the potential PSII activity decreased in cultures pre‐grown under 100 µE m−2 s−1 and incubated under 420 µE m−2 s−1. This indicates that cultures grown under higher light experience irreversible inhibition of PSII in addition to reversible down regulation. High dissolved O2 content during the oxygen evolving stage of sulfur deprivation has a negative regulatory role on PSII activity. To increase hydrogen production by C. reinhardtii pre‐grown under 100 µE m−2 s−1, cultures were incubated under elevated PFD and decreased oxygen pressure during the oxygen evolving stage. These cultures reproducibly reached anaerobic stage, accumulated significant quantities of starch and produced significant quantities of H2. It was found that elevation of pH from 7.4 to 7.7 during the oxygen producing stage of sulfur deprivation led to a significant increase of accumulated starch. Thus, control of pH during sulfur deprivation is a possible way to further optimize hydrogen production by photoautotrophic cultures. Biotechnol. Bioeng. 2009;102: 1055–1061.

New experimental approach to the estimation of rate of electron transfer from the primary to secondary acceptors in the photosynthetic electron transport chain of purple bacteria.

A method for calculating the rate constant (KA1A2) for the oxidation of the primary electron acceptor (A1) by the secondary one (A2) in the photosynthetic electron transport chain of purple bacteria is proposed. The method is based on the analysis of the dark recovery kinetics of reaction centre bacteriochlorophyll (P) following its oxidation by a short single laser pulse at a high oxidation-reduction potential of the medium. It is shown that in Ectothiorhodospira shaposhnikovii there is little difference in the value of KA1A2 obtained by this method from that measured by the method of Parson ((1969) Biochim, Biophys. Acta 189, 384-396), namely: (4.5 +/- 1.4)-10(3) s-1 and (6.9 +/- 1.2)-10(3) s-1, respectively. The proposed method has also been used for the estimation of the KA1A2 value in chromatophores of Rhodospirillum rubrum deprived of constitutive electron donors which are capable of reducing P+ at a rate exceeding this for the transfer of electron from A1 to A2. The method of Parson cannot be used in this case. The value of KA1A2 has been found to be (2.7 +/- 0.8)-10(3) s-1. The activation energies for the A1 to A2 electron transfer have also been determined. They are 12.4 kcal/mol and 9.9 kcal/mol for E. shaposhnikovii and R. rubrum, respectively.

THE ROLE OF MEMBRANE-BOUND PORPHYRIN-TYPE COMPOUND AS ENDOGENOUS SENSITIZER IN PHOTODYNAMIC DAMAGE TO YEAST PLASMA MEMBRANES

The effect of visible light (400-600 nm) on Candida guilliermondii and Saccharomyces cerevisiae was studied, and irradiation fluences killing the yeast cells without exogenous sensitizers were determined. The lethal effects are strongly oxygen-dependent, suggesting the involvement of photodynamic reactions mediated by endogenous sensitizer(s). Repair-deficient strains of S. cerevisiae show the same photosensitivity as the wild-type strain indicating that visible light does not photosensitize repairable DNA lesions. As was demonstrated using the microfluorometric method with the fluorochrome primulin, photodestruction of plasma membrane permeability barriers is important for yeast cell lethality. Visible light at cell-killing fluences induces lipid peroxidation in plasma membrane ghosts isolated from C. guilliermondii. Data obtained suggest the important role of singlet oxygen photogenerated by endogenous sensitizer(s) in initiating oxidative reactions. A spectrofluorometric analysis of the plasma membrane ghosts revealed one compound fluorescent in the visible spectral region at 683 nm. Its fluorescence excitation and absorption spectra have structures typical for porphyrins. The plasma membrane-bound porphyrin-like compound is different in some fluorescence properties from mitochondrial porphyrins. Estimation of its amount gives a value of 0.1 nmol porphyrin per milligram of protein of the plasma membrane ghosts. This porphyrin-type compound is considered to be the most probable candidate for the role of the sensitizer in photodynamic damage to yeast plasma membrane and cell inactivation by visible light.

Hydrogen photoproduction in green algae Chlamydomonas reinhardtii under magnesium deprivation

The effect of Mg-deprivation on green algae Chlamydomonas reinhardtii was studied. It resulted in the decrease of photosynthetic activity, increased respiration and accumulation of starch. After 35 hours anaerobic conditions were established and sustained H2 evolution (>7 days) was detected.

Probing the fluorescence emission kinetics of the photosynthetic apparatus of Rhodopseudomonas sphaeroides, strain 1760-1, on a picosecond pulse fluorometer

Abstract Using the pulse picosecond fluorometric technique the fluorescence properties of intact cells, isolated chromatophores and photosynthetic reaction centres were studied in bacteria Rhodopseudomonas sphaeroides , strain 1760-1. The fluorescent emission from reduced reaction centres excited by 694.3 nm light has a biphasic character, the lifetimes of the components being τ 1 = 15±8 ps and τ 2 = 250 ps. The faster component, τ 1 , contributes to the integral fluorescence in the long wavelength region. It disappears with oxidation of the reaction centres and is attributed to photoactive bacteriochlorophyll P870. The slow component, τ, is apparently due to both bacteriochlorophyll P800 and bacteriopheophytin. The fluorescence from intact cells exhibits a monophasic pattern and decays with τ = 200 ps. The fluorescence emitted by chromatophores comprises two components with τ 3 = 200 ps and τ 4 = 4200 ps. The duration of fluorescence τ 3 increases to its maximum of 500–550 ps, as P870 is oxidized chemically or photochemically, while τ 4 remains unchanged. The fluorescence with a lifetime of 200 ps was ascribed to the photosystem and the 4200-ps fluorescence to bacteriochlorophyll which had lost its functional links with the photosystem. The rise time of the fluorescence emitted by chromatophores varies from 60 or 70 ps to 350 ps depending on the wavelength of the exciting light and the recorded spectral region. On the basis of our findings the rate for energy migration was estimated to be 10 9 s −1 .

The Relationship between the Photosystem 2 Activity and Hydrogen Production in Sulfur Deprived Chlamydomonas reinhardtiiCells

Previous studies showed that, after 1.5–2 days of adaptation to inorganic S deprivation, the unicellular alga C. reinhardtii is capable of maintaining an intense hydrogen production for several days under actinic light illumination [3, 4]. As was demonstrated earlier, incubation of cells in S-deprived medium during the first 24 h results in a progressive reduction of the photosynthetic rate due to reversible inactivation of PS2 by 85–90% [5]. When the rate of photosynthetic production of O 2 declines below the rate of cell respiration, the cell culture turns into an anaerobic state followed by the onset of H 2 production [3]. However, the actual mechanisms responsible for the change in the PS2 activity during the cell’s transition into the anaerobic state and the role of PS2 in H 2 production in S-deprived cells need to be studied in more detail.