Acetate Metabolism in the Purple Non-sulfur Bacterium Rhodobacter capsulatus.

The purple non-sulfur bacterium Rhodobacter capsulatus B10 can grow on acetate as the sole carbon source under photoheterotrophic conditions. It is known that the bacterium can use the glyoxylate cycle and, in addition to it, or alternatively to it, an unknown pathway for acetate assimilation. We analyzed the genetic potential for functioning of additional metabolic pathways of oxaloacetic acid (OAA) pool replenishment in R. capsulatus. Using published microarray data of more than 4000 transcripts of genes for R. capsulatus, the genes necessary for acetate assimilation were analyzed. The results of the analysis showed the presence of all genes necessary for functioning of the ethylmalonyl-CoA pathway, and also a combination of pathways of formation of pyruvic acid/phosphoenol pyruvate (PA/PEP) (from acetyl-CoA and formate, from acetyl-CoA and CO2, as well as from 3-phosphoglyceric acid formed in the Calvin-Benson cycle) with their subsequent carboxylation. Using expression analysis, we showed that OAA pool replenishment on acetate medium could be achieved via a combination of PA/PEP synthesis from Calvin-Benson cycle intermediates and their carboxylation (with participation of pyruvate carboxylase, two reversible malate dehydrogenases (decarboxylating) and PEP-carboxykinase) to tricarboxylic acid cycle intermediates, the glyoxylate cycle, and a modified ethylmalonyl-CoA pathway in R. capsulatus under these experimental conditions. It was found that analogs of ethylmalonyl-CoA pathway enzymes exist. These enzymes differ in their specificity for S-enantiomers.

Modeling three-dimensional structure of two closely related Ni-Fe hydrogenases.

The results of homology modeling of HydSL, a NiFe-hydrogenase from purple sulfur bacterium Thiocapsa roseopersicina BBS, and deep-water bacterium Alteromonas macleodii deep ecotype are presented in this work. It is shown that the models have larger confidence level than earlier published ones; full-size models of these enzymes are presented for the first time. The C-end fragment of small subunit of T. roseopersicina hydrogenase is shown to have random orientation in relation to the main protein globule. The obtained models of this enzyme have a large number of ion pairs, as well as thermostable HydSL hydrogenase from Allochromatium vinosum, in contrast to thermostable HydSL hydrogenase from Alt. macleodii and thermolabile HydAB hydrogenase from Desulfovibrio vulgaris. The possible determinant of oxygen stability of studied hydrogenases could be the lack of several intramolecular tunnels. Hydrophobic and electrostatic surfaces were mapped in order to find out possible pathways of coupling hydrogenase to electron-transferring chains, as well as methods for construction of artificial photobiohydrogen-producing systems.

Development of bacteriochlorophyll a-based near-infrared photosensitizers conjugated to gold nanoparticles for photodynamic therapy of cancer.

We report the synthesis and characterization of a new sulfur-containing derivative of bacteriochlorophyll a. The latter was isolated from biomass of the nonsulfur purple bacterium Rhodobacter capsulatus strain B10. The developed photosensitizer is N-aminobacteriopurpurinimide with an exocyclic amino group acylated with a lipoic acid moiety, which is a biogenic substance that acts as a cofactor of the pyruvate dehydrogenase and α-ketoglutarate dehydrogenase complexes in the body. The disulfide moiety of lipoic acid confers the compound aurophilicity, thus allowing its conjugation with gold nanoparticles (NP-Au) via S-Au bonds. The shape and the size of the resulting nanoconjugate with immobilized photosensitizer (PS-Au) were assessed by dynamic light scattering and transmission electron microscopy. The conjugated nanoparticles are spherical with hydrodynamic diameter of 100-110 nm. The PS-Au conjugate absorbs light at 824 nm and emits strong fluorescence at 830 nm, which allowed in vivo study of its dynamic biodistribution in rats bearing sarcoma M-1. Compared to the free photosensitizer, PS loaded on the gold nanoparticles (PS-Au) showed extended circulation time in the blood and enhanced tumor uptake due to nonspecific passive targeting when the drug accumulates in tumor sites through the leaky tumor neovasculature and does not return to the circulation.

Interaction of HydSL hydrogenase from the purple sulfur bacterium Thiocapsa roseopersicina BBS with methyl viologen and positively charged polypeptides.

The effect of polypeptides having different charge on the activity of Thiocapsa roseopersicina HydSL hydrogenase was studied. Strong inhibition was shown for poly-L-lysine bearing positive charge. The inhibition was reversible and competitive to methyl viologen, an electron acceptor, in the reaction of hydrogen oxidation catalyzed by the hydrogenase. Peptides carrying less positive charge had weaker inhibiting effect, while neutral and negatively charged peptides did not inhibit the hydrogenase. Molecular docking of poly-L-lysine to T. roseopersicina hydrogenase showed strong affinity of this polypeptide to the acceptor-binding site of the enzyme. The calculated binding constant is close to the experimentally measured value (Ki = 2.1 µM).

[Coupled biological hydrogen-producing systems: a review].

Possible ways for combining various biological processes of biohydrogen production are described. Some of these processes are being intensively studied now, whereas others are theoretically feasible, but as yet have not been studied. A special focus is on the factors that influence the efficiency of coupled systems.

The relationship between photosystem II regulation and light-dependent hydrogen production by microalgae.

Certain microalgae species are capable of light-dependent hydrogen production under conditions of dark anaerobic incubation or nutrient deprivation. From the biotechnological point of view, this phenomenon is a process of synthesizing the energy carrier H2 while consuming light energy. Here, we overview the functional connection between the photosynthetic machinery and light-dependent hydrogen production and assess the physiological significance of this process. We characterize events involved in PSII downregulation, as well as the relationship between PSII regulation mechanisms and hydrogen generation. We suggest that the light-dependent hydrogen production forms part and parcel of the sophisticated regulatory network ensuring adaptation of microalgae to such environmental stresses as anaerobiosis or nutrient deprivation.

Porous amorphous nitinol synthesized by argon injection: a molecular dynamics study.

Porous crystalline nitinol is widely applied in various fields of science and technology due to the unique combination of physical and mechanical properties as well as biocompatibility. Porous amorphous nitinol is characterized by improved mechanical properties compared to its crystalline analogues. Moreover, this material is more promising from the point of view of fundamental study and practical application. The production of porous amorphous nitinol is a difficult task requiring rapid cooling protocol and optimal conditions to form a stable porous structure. In the present work, based on the results of molecular dynamics simulations, we show that porous nitinol with the amorphous matrix can be obtained by injection of argon into a liquid melt followed by rapid cooling of the resulting mixture. We find that the porosity of the system increases exponentially with increasing fraction of injected argon. It has been established that the system should contain about∼18%-23% argon for obtain an open porous structure, while the system is destroyed by overheated inert gas when the argon fraction is more than∼23%. It is shown that the method of argon injection makes it possible to obtain a highly porous system with the porosity∼70% consisting the spongy porous structure similar to aerogels and metallic foams.

[Nitrogen-fixing cyanobacteria: producents of hydrogen].

The review sums up recent data on the key enzymes involved in hydrogen release. Hydrogen release by cyanobacteria is due to the effects of nitrogenases and hydrogenases. Cyanobacteria hold important practical implications as transformers of solar energy into molecular hydrogen viewed as a fuel. Avenues of further research in the field are discussed.

[Synthesis of bacteriochlorophyll a by the purple nonsulfur bacterium Rhodobacter capsulatus].

The ability of purple nonsulfur bacteria Rhodobacter capsulatus B10 to synthesize bacteriochlorophyll under phototrophic and dark conditions was studied. The modes for cultivation in the dark with oxygen limitation in a continuous culture at D = 0.1 h(-1) were selected. The yield of biomass reached 20 g/l; the bacteriochlorophyll a output of the process amounted to 16.6 mg/l h(-1).

The dependence of algal H2 production on Photosystem II and O2 consumption activities in sulfur-deprived Chlamydomonas reinhardtii cells.

Chlamydomonas reinhardtii cultures, deprived of inorganic sulfur, undergo dramatic changes during adaptation to the nutrient stress [Biotechnol. Bioeng. 78 (2002) 731]. When the capacity for Photosystem II (PSII) O(2) evolution decreases below that of respiration, the culture becomes anaerobic [Plant Physiol. 122 (2000) 127]. We demonstrate that (a) the photochemical activity of PSII, monitored by in situ fluorescence, also decreases slowly during the aerobic period; (b) at the exact time of anaerobiosis, the remaining PSII activity is rapidly down regulated; and (c) electron transfer from PSII to PSI abruptly decreases at that point. Shortly thereafter, the PSII photochemical activity is partially restored, and H(2) production starts. Hydrogen production, which lasts for 3-4 days, is catalyzed by an anaerobically induced, reversible hydrogenase. While most of the reductants used directly for H(2) gas photoproduction come from water, the remaining electrons must come from endogenous substrate degradation through the NAD(P)H plastoquinone (PQ) oxido-reductase pathway. We propose that the induced hydrogenase activity provides a sink for electrons in the absence of other alternative pathways, and its operation allows the partial oxidation of intermediate photosynthetic carriers, including the PQ pool, between PSII and PSI. We conclude that the reduced state of this pool, which controls PSII photochemical activity, is one of the main factors regulating H(2) production under sulfur-deprived conditions. Residual O(2) evolved under these conditions is probably consumed mostly by the aerobic oxidation of storage products linked to mitochondrial respiratory processes involving both the cytochrome oxidase and the alternative oxidase. These functions maintain the intracellular anaerobic conditions required to keep the hydrogenase enzyme in the active, induced form.

[Hydrogen in metabolism of purple bacteria and prospects for practical applications].

Purple bacteria are able to use H2 for photoautotrophic, photomixotrophic, and chemoautotrophic growth, exhibiting high metabolic lability. Depending on the type of metabolism, hydrogen may be consumed with release of energy and/or reductive equivalents. Purple bacteria may also release H2 as a terminal electron acceptor or in the course of dinitrogen fixation. Thus, hydrogen metabolism in purple bacteria is diverse; these bacteria are often used as models for investigation of the metabolic traits and interrelation of the metabolic pathways involving molecular hydrogen. In this review, the present-day state of investigation of hydrogen metabolism in purple bacteria is reflected and its possible practical applications are discussed. Nitrogenase and hydrogenase, the major key enzymes of hydrogen metabolism, are discussed in brief. A generalized scheme of H2 role in the metabolism of purple bacteria is presented. Experimental approaches for investigation of the rates of hydrogen production are discussed. Immobilized systems are noted as the most promising approach for development of model systems for hydrogen production.

Effect of O2, H2 and redox potential on the activity and synthesis of hydrogenase 2 in Escherichia coli.

The aim of this work was to study the influence of O2 with special emphasis on low oxygen tension, the effect of H2 under various conditions of oxygen tension and the influence of the redox potential in the growth medium on hydrogenase 2 of Escherichia coli. The hydrogenase activity and the content of the large (HybC) and small (HybO) subunits of hydrogenase 2 were compared during turbidostat cultivation in a wild strain and mutant HDK103 lacking hydrogenases 1 and 3. No hydrogenase 2 activity in the mutant HDK103 was observed under aerobic conditions, but it was maximal under anaerobic conditions and half-maximal at an oxygen tension of approximately 4 mbar as is common for enzymes of anaerobic respiration. The content of hydrogenase 2 in both the strains was maximal under anaerobic conditions. In the wild strain, H2 addition enhanced hydrogenase activity and the HybO content under microaerobic conditions only. Under anaerobic conditions endogenous H2 production hindered this effect. Under aerobic conditions, the 02-related negative effect seemed to dominate over the H2-related positive effect. By contrast, in the mutant HDK103, hydrogen influenced neither hydrogenase 2 activity nor its content. A possible role of hydrogenase I in the response of hydrogenase 2 to hydrogen is discussed. Under conditions of different O2 tension, hydrogenase activity in both strains correlated inversely with the value of the redox potential of the medium. The presence of H2 changed this dependence. Thus, the value of the redox potential itself is not a controlling factor for hydrogenase 2.

H2 consumption by Escherichia coli coupled via hydrogenase 1 or hydrogenase 2 to different terminal electron acceptors.

Hydrogen uptake in the presence of various terminal electron acceptors was examined in Escherichia coli mutants synthesizing either hydrogenase 1 or hydrogenase 2. Both hydrogenases mediated nitrate-dependent H2 consumption but neither of them was coupled with nitrite. Unlike hydrogenase 2, hydrogenase 1 demonstrated poor activity with electron acceptors of low midpoint redox potential. Oxygen-linked H2 uptake via hydrogenase 1 was observed over a wide range of air concentrations. Hydrogenase 2 catalyzed this reaction only at low air concentrations. Thus, hydrogenase 1 works in cells at higher redox potential, being more tolerant to oxygen than hydrogenase 2.

[New photosensitizers of bacteriochlorin series for photodynamic cancer therapy]. / Novye fotosensibilizatory bakteriokhlorinovogo riada dlia fotodinamicheskoi terapii raka.

New derivatives of bacteriochlorophyll a bearing an extra glutarimide exocycle were synthesized, and their reactivity was studied. Acetyl group in 3-acetyl-2,7,12,18-tetramethyl-8-ethyl-13,15- dicarboxy-17-carboxyethyl-7,8,17,18-tetrahydroporphyrin (bacteriochlorin p) was chemically modified into alpha-hydroxyethyl and vinyl groups. A simple method of preparation of vinylbacteriopurpurin esters under the catalysis by p-toluenesulfonic acid was proposed. The resulting compounds exhibit a high adsorption in the visible and near IR areas of electronic spectra, a reasonable stability, and amphiphilic properties and, therefore, may be regarded as promising photosensitizers for the photodynamic cancer therapy.

[Mass-energy balance of the growth of phototropic purple bacteria]. / Material'no-énergeticheskii balans rosta fototrophnykh purpurnykh bakterii.

The principles of the theory of mass-energy balance of the growth of cellular populations are described. Based on this theory, the effect of biochemical parameters of cell metabolism on the efficiency of phototrophic growth of bacterial culture was studied. The metabolism of phototrophic bacteria was subdivided into constructive and energetic partial metabolisms. The stoichiometric coefficients describing the energetics of the two metabolism types were calculated. An equation system of the mass-energy balance for the flows of reductivity, high-energy bonds, and protons--carriers of the transmembrane electrochemical potential was derived. Equations for biomass quantum yield were obtained. The calculated yield values are in agreement with experimental data.

[Laboratory photobioreactors]. / Laboratornye fotobioreaktory.

Growing phototrophic microorganisms consume the light energy. These microorganisms most often suffer from light deficiency because of exponential decrease in the energy of light passing through an absorbing medium. Therefore, effective distribution of light within the cultures is needed for their intensive cultivation. This is possible in special devices called photobioreactors. The photobioreactors described in the literature are classified into several types according to their geometric features. Their advantages and drawbacks are analyzed. Criteria currently used for comparing various photobioreactors are specified.

Demonstration of hydrogenase electrode operation in a bioreactor.

This work describes the first step towards combination of the bioreactor with a starch-degrading microbial consortium and hydrogenase electrode (HE) in one unit for electricity generation. For this purpose, the bioreactor for microbial fermentation was designed with a set of electrodes (pH-sensor, Ag|AgCl reference electrode, Pt-electrode, and HE) inside the bioreactor. Potentials of all electrodes and H(2) accumulation were monitored in the system under the precise pH control. Results obtained with the hydrogen-producing microbial consortium indicated that HE generates the potential equal to the H(2)|2H(+) equilibrium potential. Furthermore, HE was able to catalyze the current generation (200 µA) by consuming H(2) gas produced in the microbial consortium from starch. After 220 h of operation, HE retained at least 81% of the initial activity. Calculations of carbon balance indicated that fermentation products were similar in microbial cells without HE and with HE generating the current due to H(2) consumption.

Measuring the pH dependence of hydrogenase activities.

The pH dependences of activities of homogenous hydrogenases of Thiocapsa roseopersicina and Desulfomicrobium baculatum in the reaction of hydrogen uptake in solution in the presence of benzyl viologen and the pH dependences of catalytic currents of hydrogen oxidation by electrodes on which these hydrogenases were immobilized were compared. Maximal activities of the hydrogenases from T. roseopersicina and D. baculatum in the reaction hydrogen uptake in solution were observed at pH 9.5 and 8.5, respectively. However, the steady-state current caused by catalytic uptake of hydrogen was maximal for the T. roseopersicina hydrogenase-containing electrode at pH 5.5-6.5 under overvoltage of 30-60 mV, whereas for electrodes with D. baculatum hydrogenase it was maximal at pH 6.0-6.5. Analysis of these data suggests that pH-dependent changes in the hydrogenase activities in solution during hydrogen uptake are due not only to the effect of proton concentration on the enzyme conformation or protonation of certain groups of the enzyme active center, but they are rather indicative of changes in free energy of the reaction accompanying changes in pH.

[The mechanism of acetate assimilation in purple nonsulfur bacteria lacking the glyoxylate pathway: acetate assimilation in Rhodobacter sphaeroides cells].

The mechanism of acetate assimilation in the purple nonsulfur bacterium Rhodobacter sphaeroides, which lacks the glyoxylate pathway, is studied. It is found that the growth of this bacterium in batch and continuous cultures and the assimilation of acetate in cell suspensions are not stimulated by bicarbonate. The consumption of acetate is accompanied by the excretion of glyoxylate and pyruvate into the medium, stimulated by glyoxylate and pyruvate, and inhibited by citramalate. The respiration of cells in the presence of acetate is stimulated by glyoxylate, pyruvate, citramalate, and mesaconate. These data suggest that the citramalate cycle may function in Rba. sphaeroides in the form of an anaplerotic pathway instead of the glyoxylate pathway. At the same time, the low ratio of fixation rates for bicarbonate and acetate exhibited by the Rba. sphaeroides cells (approximately 0.1), as well as the absence of the stimulatory effect of acetate on the fixation of bicarbonate in the presence of the Calvin cycle inhibitor iodoacetate, suggests that pyruvate synthase is not involved in acetate assimilation in the bacterium Rba. sphaeroides.