K. N. Timofeev

H2O2-induced inhibition of photosynthetic O2 evolution by Anabaena variabilis cells.

Hydrogen peroxide inhibits photosynthetic O2 evolution. It has been shown that H2O2 destroys the function of the oxygen-evolving complex (OEC) in some chloroplast and Photosystem (PS) II preparations causing release of manganese from the OEC. In other preparations, H2O2 did not cause or caused only insignificant release of manganese. In this work, we tested the effect of H2O2 on the photosynthetic electron transfer and the state of OEC manganese in a native system (intact cells of the cyanobacterium Anabaena variabilis). According to EPR spectroscopy data, H2O2 caused an increase in the level of photooxidation of P700, the reaction centers of PS I, and decreased the rate of their subsequent reduction in the dark by a factor larger than four. Combined effect of H2O2, CN–, and EDTA caused more than eight- to ninefold suppression of the dark reduction of P700+. EPR spectroscopy revealed that the content of free (or loosely bound) Mn2+ in washed cyanobacterial cells was ∼20% of the total manganese pool. This content remained unchanged upon the addition of CN– and increased to 25-30% after addition of H2O2. The content of the total manganese decreased to 35% after the treatment of the cells with EDTA. The level of the H2O2-induced release of manganese increased after the treatment of the cells with EDTA. Incubation of cells with H2O2 for 2 h had no effect on the absorption spectra of the photosynthetic pigments. More prolonged incubation with H2O2 (20 h) brought about degradation of phycobilins and chlorophylla and lysis of cells. Thus, H2O2 causes extraction of manganese from cyanobacterial cells, inhibits the OEC activity and photosynthetic electron transfer, and leads to the destruction of the photosynthetic apparatus. H2O2 is unable to serve as a physiological electron donor in photosynthesis.

Role of chloroplast photosystems II and I in apoptosis of pea guard cells.

We investigated the CN–-induced apoptosis of guard cells in epidermal peels isolated from pea (Pisum sativum L.) leaves. This process was considerably stimulated by illumination and suppressed by the herbicides DCMU (an inhibitor of the electron transfer between quinones QA and QB in PS II) and methyl viologen (an electron acceptor from PS I). These data favor the conclusion drawn by us earlier that chloroplasts are involved in the apoptosis of guard cells. Pea mutants with impaired PS I (Chl-5), PS II (Chl-I), and PS II + PS I (Xa-17) were tested. Their lesions were confirmed by the ESR spectra of Signal I (oxidized PS I reaction centers) and Signal II (oxidized tyrosine residue YD in PS II). Destruction of nuclei (a symptom of apoptosis) and their consecutive disappearance in guard cells were brought about by CN– in all the three mutants and in the normal pea plants. These results indicate that the light-induced enhancement of apoptosis of guard cells and its removal by DCMU are associated with PS II function. The effect of methyl viologen preventing CN–-induced apoptosis in wild-type plants was removed or considerably decreased upon the impairment of the PS II and/or PS I activity.

The primary reactions in the protochlorophyll(ide) photoreduction as investigated by optical and ESR spectroscopy

It has been found that at low temperatures (77K–153K) a long-lived (at these temperatures) singlet ESR signal induced by intensive light appears in etiolated leaves of plants and in model systems including both the monomeric and aggregated protochlorophyll.Comparison of the results of ESR, fluorescence and absorption spectra measurements made it possible to suggest that at the initial stages of the protochlorophyll(ide) photoreduction process at least two paramagnetic non-fluorescent intermediates are formed, one of which seems to be identical to the previously found intermediate with absorption maximum at 690 nm. On the strength of the obtained results a conclusion can be drawn that photoreduction of the semi-isolated double-c=c-bond of the chlorophyll precursor molecule in etiolated leaves and in model systems is actualized via at least two stages of free radicals formation. A scheme of the primary reactions of chlorophyllide biosynthesis has been proposed.

Participation of Free Radicals in Photoreduction of Protochlorophyllide to Chlorophyllide in an Artificial Pigment–Protein Complex

The primary stages of protochlorophyllide phototransformation in an artificially formed complex containing heterologously expressed photoenzyme protochlorophyllide-oxidoreductase (POR), protochlorophyllide, and NADPH were investigated by optical and ESR spectroscopy. An ESR signal (g = 2.002; H = 1 mT) appeared after illumination of the complex with intense white light at 77 K. The ESR signal appeared with simultaneous quenching of the initial protochlorophyllide fluorescence, this being due to the formation of a primary non-fluorescent intermediate. The ESR signal disappeared on raising the temperature to 253 K, and a new fluorescence maximum at 695 nm belonging to chlorophyllide simultaneously appeared. The data show that the mechanism of protochlorophyllide photoreduction in the complex is practically identical to the in vivo mechanism: this includes the formation of a short-lived non-fluorescent free radical that is transformed into chlorophyllide in a dark reaction.

Production of reactive oxygen species in decoupled, Ca2+-depleted PSII and their use in assigning a function to chloride on both sides of PSII

Extraction of Ca2+ from the oxygen-evolving complex of photosystem II (PSII) in the absence of a chelator inhibits O2 evolution without significant inhibition of the light-dependent reduction of the exogenous electron acceptor, 2,6-dichlorophenolindophenol (DCPIP) on the reducing side of PSII. The phenomenon is known as “the decoupling effect” (Semin et al. Photosynth Res 98:235–249, 2008). Extraction of Cl− from Ca2+-depleted membranes (PSII[–Ca]) suppresses the reduction of DCPIP. In the current study we investigated the nature of the oxidized substrate and the nature of the product(s) of the substrate oxidation. After elimination of all other possible donors, water was identified as the substrate. Generation of reactive oxygen species HO, H2O2, and O2·−, as possible products of water oxidation in PSII(–Ca) membranes was examined. During the investigation of O2·− production in PSII(–Ca) samples, we found that (i) O2·− is formed on the acceptor side of PSII due to the reduction of O2; (ii) depletion of Cl− does not inhibit water oxidation, but (iii) Cl− depletion does decrease the efficiency of the reduction of exogenous electron acceptors. In the absence of Cl− under aerobic conditions, electron transport is diverted from reducing exogenous acceptors to reducing O2, thereby increasing the rate of O2·− generation. From these observations we conclude that the product of water oxidation is H2O2 and that Cl− anions are not involved in the oxidation of water to H2O2 in decoupled PSII(–Ca) membranes. These results also indicate that Cl− anions are not directly involved in water oxidation by the Mn cluster in the native PSII membranes, but possibly provide access for H2O molecules to the Mn4CaO5 cluster and/or facilitate the release of H+ ions into the lumenal space.

Role of NAD(P)H:quinone oxidoreductase encoded by drgA gene in reduction of exogenous quinones in cyanobacterium Synechocystis sp. PCC 6803 cells

Insertion mutant Ins2 of the cyanobacterium Synechocystis sp. PCC 6803, lacking NAD(P)H:quinone oxidoreductase (NQR) encoded by drgA gene, was characterized by higher sensitivity to quinone-type inhibitors (menadione and plumbagin) than wild type (WT) cells. In photoautotrophically grown cyanobacterial cells more than 60% of NADPH:quinone-reductase activity, as well as all NADPH:dinoseb-reductase activity, was associated with the function of NQR. NQR activity was observed only in soluble fraction of cyanobacterial cells, but not in membrane fraction. The effects of menadione and menadiol on the reduction of Photosystem I reaction center (P700+ ) after its photooxidation in the presence of DCMU were studied using the EPR spectroscopy. The addition of menadione increased the rate of P700+ reduction in WT cells, whereas in Ins2 mutant the reduction of P700+ was strongly inhibited. In the presence of menadiol the reduction of P700+ was accelerated both in WT and Ins2 mutant cells. These data suggest that NQR protects the cyanobacterial cells from the toxic effect of exogenous quinones by their reduction to hydroquinones. These data may also indicate the probable functional homology of Synechocystis sp. PCC 6803 NQR with mammalian and plant NAD(P)H:quinone oxidoreductases (DT-diaphorases).

Effects of oxygen on the dark recombination between photoreduced secondary quinone and oxidized bacteriochlorophyll in Rhodobacter sphaeroides reaction centers

The influence of duration of exposure to actinic light (from 1 sec to 10 min) and temperature (from 3 to 35°C) on the temporary stabilization of the photomobilized electron in the secondary quinone acceptor (QB) locus of Rhodobacter sphaeroides reaction centers (RC) was studied under aerobic or anaerobic conditions. Optical spectrophotometry and ESR methods were used. The stabilization time increased significantly upon increasing the exposure duration under aerobic conditions. The stabilization time decreased under anaerobic conditions, its dependence on light exposure duration being significantly less pronounced. Generation of superoxide radical in photoactivated aerobic samples was revealed by the ESR method. Possible interpretation of the effects is suggested in terms of interaction between the semiquinone QB with oxygen, the interaction efficiency being determined by the conformational transitions in the structure of RC triggered by actinic light on and off.

Reduction of photosystem I reaction center in DrgA mutant of the cyanobacterium Synechocystis sp. PCC 6803 lacking soluble NAD(P)H:quinone oxidoreductase.

Photoautotrophically grown cells of the cyanobacterium Synechocystis sp. PCC 6803 wild type and the Ins2 mutant carrying an insertion in the drgA gene encoding soluble NAD(P)H:quinone oxidoreductase (NQR) did not differ in the rate of light-induced oxygen evolution and Photosystem I reaction center (P700+) reduction after its oxidation with a white light pulse. In the presence of DCMU, the rate of P700+ reduction was lower in mutant cells than in wild type cells. Depletion of respiratory substrates after 24 h dark-starvation caused more potent decrease in the rate of P700+ reduction in DrgA mutant cells than in wild type cells. The reduction of P700+ by electrons derived from exogenous glucose was slower in photoautotrophically grown DrgA mutant than in wild type cells. The mutation in the drgA gene did not impair the ability of Synechocystis sp. PCC 6803 cells to oxidize glucose under heterotrophic conditions and did not impair the NDH-1-dependent, rotenone-inhibited electron transfer from NADPH to P700+ in thylakoid membranes of the cyanobacterium. Under photoautotrophic growth conditions, NADPH-dehydrogenase activity in DrgA mutant cells was less than 30% from the level observed in wild type cells. The results suggest that NQR, encoded by the drgA gene, might participate in the regulation of cytoplasmic NADPH oxidation, supplying NADP+ for glucose oxidation in the pentose phosphate cycle of cyanobacteria.

Superoxide Mediated Chlorophyll Allomerization in a Dimethyl Sulphoxide-Water Mixture

The interaction of chlorophyll a with superoxide anion was studied in an alkaline DMSO-water system. It was found that O2(-.), directly or via HO2., produces the chlorophyll enolate-ion (Molishs intermediate) that is oxidized to Mg-chlorine(s). The allomerization reaction was found to be inhibited by superoxide dismutase. A possible participation of oxygen radicals in chlorophyll degradation in plants is discussed.

On the mechanisms of photodynamic effect of sensitizers and improvement of methods of their primary selection for photodynamic antimicrobial therapy.

In the past years, photodynamic therapy (PDT) is widely used in oncology and antimicrobial therapy [1, 2]. This method is based on cell damage by reactive oxygen species (ROS) that are generated by photosensitizers in the photoexcited state. A broad spectrum of photosensitizers, including predominantly porphyrins and phthalocyanine, has been created [1, 2]. Primary selection of new promising compounds requires rapid model cell methods (e.g., bioluminescent bacterial sensors) and new technologies of their usage. Numerous studies showed that approximately 50% inhibition of luminescence of biosensors by toxicants ( Öë 50 ) correlates with LD 50 for animals [3]. Ecolum test systems based on natural luminous species of marine bacteria and the genetically transformed Escherichia coli strain K12 TG1, into which the lux operon of different species of luminous bacteria was cloned, were created at the Department of Microbiology, Moscow State University [4]. We were the first to use the Ecolum-08 test system, which is based on the genetically transformed E. coli strain K12 TG1 containing the lux operon from soil entomopathogenic luminous gram-negative bacteria Photorhabdus luminescens strain ZM1, to assess the efficiency of photosensitizers that are used in PDT of tumors [5, 6]. This biosensor also showed promise when selecting photosensitizers for antimicrobial PDT of various infections. It was found that cationic photosensitizers, which actively bind with negatively charged surface of bacterial cell, are more effective [7]. Interestingly, one of the key properties of effective photodynamic dyes is their ability to interact with target cells. To enhance this interaction, empiric procedures are developed, for example, the use of conjugates of photosensitizer with antibodies to bacteria [8, 9]. The development of theoretically substantiated ways of targeted delivery of photosensitizers into the nucleus of target cells is also a promising approach [10]. Novel cationic-type photosensitizers that exhibit affinity for cells are synthesized for their photodynamic destruction [7, 11]. However, a simple and rapid procedure is required for the estimation of the interaction of photosensitizers with cells.