Effects of benzyl viologen on increasing NADH availability, acetate assimilation, and butyric acid production by Clostridium tyrobutyricum.

Clostridium tyrobutyricum produces butyric and acetic acids from glucose. The butyric acid yield and selectivity in the fermentation depend on NADH available for acetate reassimilation to butyric acid. In this study, benzyl viologen (BV), an artificial electron carrier that inhibits hydrogen production, was used to increase NADH availability and butyric acid production while eliminating acetic acid accumulation by facilitating its reassimilation. To better understand the mechanism of and find the optimum condition for BV effect on enhancing acetate assimilation and butyric acid production, BV at various concentrations and addition times during the fermentation were studied. Compared with the control without BV, the addition of 1 µM BV increased butyric acid production from glucose by ∼50% in yield and ∼29% in productivity while acetate production was completely inhibited. Furthermore, BV also increased the coutilization of glucose and exogenous acetate for butyric acid production. At a concentration ratio of acetate (g/L) to BV (mM) of 4, both acetate assimilation and butyrate biosynthesis increased with increasing the concentrations of BV (0-6.25 µM) and exogenous acetate (0-25 g/L). In a fed-batch fermentation with glucose and ∼15 g/L acetate and 3.75 µM BV, butyrate production reached 55.9 g/L with productivity 0.93 g/L/h, yield 0.48 g/g, and 97.4% purity, which would facilitate product purification and reduce production cost. Manipulating metabolic flux and redox balance via BV and acetate addition provided a simple to implement metabolic process engineering approach for butyric acid production from sugars and biomass hydrolysates.

[Effect of oxidative stress inductors on the photosynthetic apparatus in cyanobacterium Synechocystis sp. PCC 6803 Prq20 mutant resistant to methyl viologen].

The damaging effect of oxidative stress inductors: methyl viologen, benzyl viologen, cumene hydroperoxide, H2O2, menadion, and high irradiance on the photosynthetic apparatus of cyanobacterium Synechocystis sp. PCC 6803 in cells of the wild type strain and the methyl viologen-resistant Prq20 mutant with the disrupted function of the regulatory gene prqR has been investigated by measuring the delayed fluorescence of chlorophyll a and the rate of CO2dependent -O2 gas exchange. It has been shown that the damage to the photosynthetic apparatus in the Prq20 mutant as compared with the wild type was less in the presence of methyl viologen and benzyl viologen. Reasons for the enhanced resistance of the photosynthetic apparatus in the mutant Prq20 to methyl viologen and benzyl viologen are discussed.

Electron donors and inhibitors of nitrate reductase from Cyanidium caldarium.

Studies on nitrate reductase (NAD(P)H:nitrate oxidoreductases EC 1.6.6.2) of Cyanidium caldarium revealed that the enzyme is inhibited by excess of electron donor, NADPH, reduced benzylviologen and FMN. Also dithionite, used to reduce benzylviologen and FMN, inactivates nitrate reductase: however, FMN at an optimal concentration and nitrate, added before the dithionite, protect the enzyme against this inactivation. Cyanide, cyanate and carbamyl phosphate inhibit the enzyme competitively with respect to nitrate, and Ki values are reported. Organic mercurials, 0.1 mM, act preferentially on NADPH activity, whereas Ag+ and Hg-2+ at the same concentration inactivate 80--90% of the benzylviologen and FMN activities. ADP is very poor inhibitor. Urea 4 M in 2 h destroys 90% of the NADPH activity and only 30% of the benzylviologen and FMN activities. The apparent Km values for NADPH, benzylviologen, FMN and nitrate have been determined.

Studies on the structural and functional organization of system II of photosynthesis. The use of trypsin as a structurally selective inhibitor at the outer surface of the thylakoid membrane.

The effect of trypsin on the photosynthetic electron transport has been investigated in the presence of various electron acceptors (benzyl viologen, p-benzo-quinone, K3[Fe(CN)6]) by measurements of flash-induced oxygen evolution and of the absorption changes at 334 nm, indicating the primary electron acceptor of System II, X 320, and at 515 nm, indicating via electrochromism the electrical potential gradient across the thylakoid membrane. It was found that the effect of trypsin is strongly dependent on the nature of the electron acceptor: (1) Oxygen evolution is completely inhibited in the presence of p-benzo-quinone, but remains nearly unaffected by K3[Fe(CN)]6. (2) The initial amplitude deltaAO of the 334 nm absorption change is insensitive to trypsin in the presence of K3[Fe(CN)6], but the absorption change is abolished if benzyl viologen is used as acceptor. (3) The initial amplitude deltaAO of the 515 nm absorption change decreases by trypsin down to 50% with K3[Fe(CN)6] and is completely suppressed with benzyl viologen. (4) In trypsinated chloroplasts, the above-mentioned activities appear to be rather insensitive to 3-(3,4-dichlorophenyl)-1,1-dimethylurea, in contrast to normal chloroplasts. On the basis of these results it is inferred that the primary electron acceptor of System II, X 320, is covered by a proteinaceous component susceptible to tryptic digestion. In addition, it is postulated that this component acts as well as an allosteric protein responsible for the regulation of the electronic interaction between X 320 and the plastoquinone pool, as for the inhibitory effect of 3-(3,4-dichlorophenyl)-1,1-dimethylurea. Various other possible effects caused by the proteinaceous shield and its modification by trypsin are discussed. The present results are in complete agreement with asymmetric membrane models postulating a zig-zag arrangement of the electron transport chain with the reducing side located towards the outer phase and the oxidizing side near the inner phase of the thylakoids.

Redox cycling of the herbicide paraquat in microglial cultures.

Mechanisms involved in paraquat neurotoxicity that selectively target nigrostriatal dopaminergic neurons remain relatively unknown. In this study, we tested the hypotheses that paraquat exposure leads to the production of reactive oxygen species (ROS) through a process of redox cycling and that microglia represent an important site for the initiation of redox cycling reactions. Addition of paraquat to N9 microglial cultures resulted in a dose- and time-dependent release of superoxide radicals. Other agents that share with paraquat the property of redox cycling, i.e., benzyl viologen and diquat, also induced a marked production of superoxide radicals by microglia. The ability of paraquat, benzyl viologen, and diquat to induce superoxide release was correlated to their one-electron reduction potentials and thus their tendency to redox cycle. Nitric oxide synthase and NADPH oxidase were identified as enzymatic sources of electrons that triggered paraquat redox cycling by microglia. Taken together, these data provide evidence in favor of a new mechanism by which microglia could play a role in oxidative injury during neurodegenerative processes. Microglial NOS and NADPH oxidase could promote the generation of ROS via the redox cycling of paraquat-like toxicants.

Genetic and physiological characterization of new Escherichia coli mutants impaired in hydrogenase activity.

The Mu dl (ApR lac) bacteriophage was used to generate mutants of Escherichia coli which were defective in formate hydrogenlyase. Three mutants were chosen for further analysis: they lacked hydrogenase (hydrogen: benzyl viologen oxidoreductase) activity, but produced normal levels of fumarate reductase activity and two- to three-fold reduced levels of benzyl viologen (BV)-dependent formate dehydrogenase activity. Two of them (hydC) were shown to contain about 4-fold reduced amounts of formate hydrogenlyase and fumarate-dependent H2 uptake activities. The third one (hydD) was totally devoid of both activities. Their insertion sites were located at 77 min on the E. coli map. Subdivision of these mutants into two classes was subsequently based on the restoration capacity of hydrogenase activity with high concentration of nickel in the growth media. Addition of 500 microM NiCl2 led to a complete recovery of hydrogenase activity, and to the concomitant restoration of normal BV-linked formate dehydrogenase, formate hydrogenlyase and fumarate-dependent H2 uptake activities in the hydC mutants. The hydD mutant was insensitive to the effect of nickel. Expression of the lac operon in hydC and hydD mutants was induced by anaerobiosis. It was not increased by the addition of formate under anaerobic conditions. The presence of nitrate resulted in slightly reduced beta-galactosidase activities in the hydC mutants, whereas those found in the hydD mutant reached only one third of the level obtained in its absence. Fumarate had no effect on both classes. Moreover, in contrast to the hydD locus, the hydC::Mu dl fusions were found to be dependent upon the positive control exerted by the nirR gene product and were totally repressed by an excess of nickel. In addition, the low levels of overall hydrogenase-dependent activities found in a nirR strain were also relieved by the presence of nickel. Our results strongly suggest that the pleiotropic regulatory gene nirR is essential for the expression of a gene (hydC) involved in either transport or processing of nickel in the cell, whose alteration leads to a loss of hydrogenase activity.

Benzyl viologen inactivation of rat liver glutamine synthetase.

Partially purified glutamine synthetase from rat liver underwent rapid inactivation upon incubation with NADH and benzyl viologen, under aerobic conditions. This in vitro inactivation was prevented by catalase or chelating-agents, which suggests that hydrogen peroxide and metal ions are involved in the process. Similar inactivation was observed when the rat liver glutamine synthetase was preincubated, under anaerobic conditions, with NADH and benzyl viologen, and hydrogen peroxide was added to the reaction mixture. A radical scavenger, histidine, partially prevents the inactivation, while hydrogen peroxide shows a low inactivation capacity when incubated without NADH. Furthermore, the fact that the inactivation can also be catalyzed by a model consisting of ferrous ions and hydrogen peroxide leads to the conclusion that hydroxyl radicals, or something with similar reactivity, are most likely produced through a Fenton reaction.

Bacterial oxidative stress substance spontaneously recyclizes to form 2-methylbutane-1,2,3,4-tetraol-1,2-cyclophospho-4-phosphate.

The cells of Corynebacterium (Brevibacterium) ammonia-genes cultivated in a medium supplemented with diquat or benzylviologen accumulate 2-methylbutane-1,2,3,4-tetraol-2,4- cyclopyrophosphate as revealed by 31P-NMR spectroscopy. On heating at 120 degrees C for 30 min the cells still maintain a substantial portion of this compound and acquire new cyclic phosphates characterized by 31P-NMR chemical shifts of +17.3 and +20 p.p.m. The +17.3 p.p.m. component was isolated from the preparation of the purified cyclopyrophosphate kept for some time at pH above 7 and it was shown to be 2-methylbutane-1,2,3,4-tetraol-1,2,- cyclophospho-4-phosphate on the grounds of two-dimensional NMR spectroscopy.

Bacteria and pesticides: a new aspect of interaction--involvement of a new biofactor.

Positively charged hydrophobic pesticides of the dipyridyl family [diquat, paraquat, benzylviologen (BV++), etc.] were shown to provoke accumulation of 2-methylbutane-1,2,3,4-tetraol-2,4- cyclopyrophosphate in the cells Corynebacterium (Brevibacterium) ammoniagenes while neutral dipyridyls were not. Hydrophobicity was also an important factor in this phenomenon. Of the other pesticides tested, only linuron was effective. BV++ also induced biosynthesis of the compound in Rhodococcus rhodochrous, Rh.ruber, Rh.sp. (Nocardia corynebacteroides). These microorganisms as well as most of the previously identified oxidative stress activated producers of this new cyclopyrophosphate were able to synthesize free radical generating compounds. The microorganisms concerned belong mainly to the order Actinomycetales.

Characterization of superoxide-stress sensing recombinant Escherichia coli constructed using promoters for genes zwf and fpr fused to lux operon.

To measure the toxicity experienced by superoxide-generating compounds, two plasmids were constructed in which the superoxide-inducible fpr and zwf promoters from Escherichia coli were fused to promoterless Vibrio fischeri luxCDABE operon present in plasmid pUCD615. The bioluminescent response of E. coli harboring these constructs was studied as a function of the toxicity and was shown to be specific for superoxide generating chemicals. The two promoters employed, fpr and zwf, responded differentially to the redox-chemicals tested. Furthermore, a DeltamarA strain bearing the fpr::luxCDABE fusion had a weaker response to paraquat (methyl viologen) than its isogenic parent strain, whereas zwf induction was not inhibited in DeltamarA or Deltarob strains. The fpr and zwf promoters were also induced by alkylating agents but were unresponsive in DeltamarA or Deltarob strains. Using optimized assay conditions, the abilities of these strains to differentially respond to superoxide stress and alkylating agents that may be present in contaminants proves them to be good biosensor candidates for monitoring toxicity.

Reversible inactivation of the O2-labile hydrogenases from Azotobacter vinelandii and Rhizobium japonicum.

Hydrogenases catalyze the reversible activation of dihydrogen. The hydrogenases from the aerobic, N2-fixing microorganisms Azotobacter vinelandii and Rhizobium japonicum are nickel- and iron-containing dimers that belong to the group of O2-labile enzymes. Exposure of these hydrogenases to O2 results in an irreversible inactivation; therefore, these enzymes are purified anaerobically in a fully active state. We describe in this paper an electron acceptor-requiring and pH-dependent, reversible inactivation of these hydrogenases. These results are the first example of an anaerobic, reversible inactivation of the O2-labile hydrogenases. The reversible inactivation required the presence of an electron acceptor. The rate of inactivation was first-order, with similar rates observed for methylene blue, benzyl viologen, and phenazine-methosulfate. The rate of inactivation was also dependent on the pH. However, increasing the pH of the enzyme in the absence of an electron acceptor did not result in inactivation. Thus, the reversible inactivation was not a result of high pH alone. The inactive enzyme could not be reactivated by H2 or other reductants at high pH. Titration of enzyme inactivated at high pH back to low pH was also ineffective at reactivating the enzyme. However, if reductants were present during this titration, the enzyme could be fully reactivated. The temperature dependence of inactivation yielded an activation energy of 44 kJ X mol-1. Gel filtration chromatography of active and inactive hydrogenase indicated that neither dissociation nor aggregation of the dimer hydrogenase was responsible for this reversible inactivation. We propose a four-state model to describe this reversible inactivation.

Methanogenesis from acetate: enrichment studies.

An acetate enrichment culture was initiated by inoculating anaerobic sludge from a mesophilic methane digestor into a mineral salts medium with calcium acetate as the sole carbon and energy source. This enrichment was maintained indefinitely by weekly transfer into medium of the same composition. A study of this enrichment disclosed an unexpected age-dependent inhibition of methanogenesis by H2 and formate which apparently differed from the inhibition by chloroform and benzyl viologen. This age-dependent inhibition indicated that microbial interactions of the mixed enrichment population may play a regulatory role in methane formation. Futhermore, stimulation of methanogenesis in the acetate enrichment by addition of yeast extract showed a nutrient limitation which indicated that syntrophic interactions leading to formation of growth factors may also occur. A model is presented to illustrate the possible interrelationships between methanogenic and nonmethanogenic bacteria in their growth and formation of methane and carbon dioxide from acetate.

Is hydrogen peroxide involved in the benzyl viologen-mediated in-vivo inactivation of rat liver glutamine synthetase?

After benzyl viologen administration to rats, a decrease in the rat liver glutamine synthetase activity was observed. An increase in the rat liver catalase activity was found concomitantly. In combination with the catalase inhibitor aminotriazole, benzyl viologen again diminished, but markedly, the rat liver glutamine synthetase activity. Moreover, partially purified glutamine synthetase from rat liver underwent rapid inactivation upon aerobic incubation with NAD(P)H and benzyl viologen. This inactivation was prevented by catalase, which suggests that the NAD(P)H/BV2+/O2-dependent system has a role in H2O2 production. Our results suggest that H2O2 is involved in the benzyl viologen-mediated in-vivo inactivation of the rat liver glutamine synthetase. In contrast, benzyl viologen alone or in combination with aminotriazole produced a significant increase of brain glutamine synthetase.

Spectroscopic characterization of triplet forming states in photosystem II.

Fluorescence and electron paramagnetic resonance (EPR) measurements have been applied to characterize chlorophyll triplet formation in the reaction center of photosystem II (PSII). A highly triplet forming state was generated in PSII membranes by chemical double reduction of the primary electron acceptor QA. In triplet forming PSII centers, the steady-state yield of chlorophyll fluorescence decreased to about 70% of the maximal fluorescence yield observed in closed PSII centers in which QA is singly reduced. The results are well interpreted in the framework of a model where the charge state of QA electrostatically controls the yield of primary charge separation [Schatz, G. H., Brock, H., & Holzwarth, A. R. (1988) Biophys. J. 54, 397-405]. Thus, high triplet yield and decreased, although still quite high, fluorescence indicate a charge-neutralized state of PSII in which QA is singly or doubly reduced and protonated or absent. The EPR signal of the triplet primary chlorophyll donor, 3P680, is suppressed by illumination at 77 K concomitant with the formation of a cationic radical (g = 2.0025-2.0027, and 0.92 mT wide) that is stable in the dark. This is attributed to the oxidation of an accessory chlorophyll (Chl) in the vicinity of P680. Electrostatic repulsion between Chl+ and P680+ is likely to prevent primary charge separation, and in turn triplet formation, providing a further example of electrostatic control of primary charge separation. The triplet P680 EPR signal is also suppressed in the presence of oxygen. This effect, which is almost completely reversible by removing the oxygen, is attributed to the interaction of triplet P680 with triplet O2.

Inhibition of photosynthetic sulfide oxidation by organic cations.

Photosynthetic sulfide oxidation by the purple sulfur bacterium Chromatium vinosum is strongly inhibited by the organic cations benzyl viologen ( BV2 +) and tetraphenylphosphonium (TPP+) at micromolar concentrations. Both are much more inhibitory at pH 8 than at pH 7. Inhibition probably results from uptake of benzyl viologen and tetraphenylphosphonium in response to an electrical potential gradient across the plasma membrane which increases in magnitude with increasing pH. Although both compounds appear to have the same mode of action, the biochemical mechanism of their inhibition remains to be determined.

Effect of benzyl viologen on the phospholipid fatty acid composition and some properties in hepatic microsomal membrane of rats.

The effect of benzyl viologen (a stimulator of free radical production in cells) on lipid composition, fluidity and enzymes involved in both polyunsaturated fatty acid biosynthesis and cholesterol metabolism was studied in liver microsomal membrane of adult rats. In viologen-treated animals, a significant decrease in the levels of free cholesterol and cholesteryl esters, accompanied to a decrease at the free cholesterol/phospholipid ratio, were observed. The levels of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase and acyl-coenzyme A: cholesterol acyltransferase (ACAT) were also lower in viologen-treated rats than in controls. Linoleic and arachidonic acids were both severely lower while docosatetraenoic, docosapentaenoic and docosahexaenoic acids were significantly higher as compared with controls. Furthermore, a decrease in monounsaturated/saturated ratio was found. In addition, the treatment evoked a depression in the fatty acid desaturation complex, with a diminish of delta 9, delta 6 and delta 5 desaturase activities in microsomal membrane. It was concluded that changes in phospholipid microsomal fatty acid and cholesterol content could be directly responsible for changes in membrane fluidity and function, and that extensive yield of docosahexaenoic acid may serve to maintain the physical characteristics of particular domains against oxidative stress caused by benzyl viologen treatment.

The effect of electron donors and acceptors on light-induced absorbance changes and photophosphorylation in Rhodospirillum rubrum chromatophores.

Light-induced difference spectra between 400 and 640 nm of Rhodospirillum rubrum chromatophores were performed in the presence and absence of exogenous electron donor/acceptor systems and compared with the chemical oxidation spectrum. The results indicate that the component previously defined as P430 is not a unique entity but rather represents different species, or a mixture of species, under various conditions. Under all conditions in which the reaction center bacteriochlorophyll is reversibly photooxidized, as indicated by the bleaching around 600 nm, it is also contributing to the absorbance increase around 430 nm. In one case, in presence of reduced dichloroindophenol and in the absence of oxygen, the photooxidation of reaction center bacteriochlorophyll is fully supressed. Under these conditions an irreversible change around 430 nm is still observed and seems to be due to the Soret band of b-type cytochrome. In the presence of reduced dichloroindophenol and absence of oxygen there is a marked inhibition of photophosphorylation. This inhibition is apparently due to the complete reduction of the cyclic electron carriers. Addition of the low potential dye benzyl viologen facilitates an almost complete recovery of the reversible photooxidation of reaction center bacteriochlorophyll as well as of photophosphorylation. These results indicate that the apparent mid-point potential of the primary electron acceptor in Rhodospirillum rubrum chromatophores is probably in the range of that of benzyl viologen (E'o = - 340 mV).