RNA synthesis during morphogenesis of the fungusMucor racemosus

Bacteroides succinogenes produces acetate and succinate as major products of carbohydrate fermentation. An investigation of the enzymes involved indicated that pyruvate is oxidized by a flavin-dependent pyruvate cleavage enzyme to acetyl-CoA and CO₂. Active CO₂ exchange is associated with the pyruvate oxidation system. Reduction of flavin nucleotides is CoASH-dependent and does not require ferredoxin. Acetyl-CoA is further metabolized via acetyl phosphate to acetate and ATP. Reduced flavin nucleotide is used to reduce fumarate to succinate by a particulate flavin-specific fumarate reductase reaction which may involve cytochrome b. Phosphoenolpyruvate (PEP) is carboxylated to oxalacetate by a GDP-specific PEP carboxykinase. Oxalacetate, in turn, is converted to malate by a pyridine nucleotide-dependent malate dehydrogenase. The organism has a NAD-dependent glyceraldehyde-3-phosphate dehydrogenase. The data suggest that reduced pyridine nucleotides generated during glycolysis are oxidized in malate formation and that the electrons generated during pyruvate oxidation are used to reduce fumarate to succinate.     

The interaction of metronidazole with electron carriers of the Azotobacter vinelandii respiratory particle fraction

Abstract Metronidazole, menadione bisulfite, and oxygen oxidized NADH- and dithionite-reduced flavin of the Azotobacter vinelandii respiratory particle fraction. The oxidation of NADH-reduced flavin by metronidazole was slow compared to the oxidations by menadione bisulfite and oxygen. Metronidazole caused spectral changes characteristic of cytochrome d oxidation, but the changes in NADH-reduced particles were slow.     

Composition of the redox environment of the endoplasmic reticulum and sources of hydrogen peroxide

The endoplasmic reticulum (ER) is a metabolically active organelle, which has a central role in proteostasis by translating, modifying, folding, and occasionally degrading secretory and membrane proteins. The lumen of the ER represents a separate compartment of the eukaryotic cell, with a characteristic proteome and metabolome. Although the redox metabolome and proteome of the compartment have not been holistically explored, it is evident that proper redox conditions are necessary for the functioning of many luminal pathways. These redox conditions are defined by local oxidoreductases and the membrane transport of electron donors and acceptors. The main electron carriers of the compartment are identical with those of the other organelles: glutathione, pyridine and flavin nucleotides, ascorbate, and others. However, their composition, concentration, and redox state in the ER lumen can be different from those observed in other compartments. The terminal oxidases of oxidative protein folding generate and maintain an “oxidative environment” by oxidizing protein thiols and producing hydrogen peroxide. ER-specific mechanisms reutilize hydrogen peroxide as an electron acceptor of oxidative folding. These mechanisms, together with membrane and kinetic barriers, guarantee that redox systems in the reduced or oxidized state can be present simultaneously in the lumen. The present knowledge on the in vivo conditions of ER redox is rather limited; development of new genetically encoded targetable sensors for the measurement of the luminal state of redox systems other than thiol/disulfide will contribute to a better understanding of ER redox homeostasis.     

Light-induced fluorescence changes in<Emphasis Type="Italic"> Phycomyces</Emphasis>: evidence for blue light-receptor associated flavo-semiquinones

Light-induced fluorescence changes (LIFCs) were detected in sporangiophores of the blue-light-sensitive fungus Phycomyces blakesleeanus (Burgeff). The LIFCs can be utilized as a spectrophotometric assay for blue-light photoreceptors and for the in vivo characterization of their photochemical primary reactions. Blue-light irradiation of sporangiophores elicited a transient decrease and subsequent regeneration of flavin-like fluorescence emission at 525 nm. The signals recovered in darkness in about 120 min. In contrast to blue light, near-UV (370 nm) caused an increase in the fluorescence emission at 525 nm. Because the LIFCs were altered in a light-insensitive madC mutant with a defective photoreceptor, the fluorescence changes must be associated with early photochemical events of the transduction chain. Action spectra for the fluorescence changes at 525 nm showed major peaks near 470 and 600 nm. Double-pulse experiments involving two consecutive pulses of either blue and near-UV, blue and red, or near-UV and red showed that the responses depended on the sequence in which the different wavelengths were applied. The results indicate a blue-light receptor with intermediates in the near-UV, blue and red spectral regions. We explain the results in the framework of a general model, in which the three redox states of the flavin photoreceptor, the oxidized flavin (Fl), the flavo-semiquinone (FlH^·), and the flavo-hydroquinone (FlH₂) are each acting as chromophores with their own characteristic photochemical primary reactions. These consist of the photoreduction of the oxidized flavin generating semiquinone, the photoreduction of the semiquinone generating hydroquinone, and the photooxidation of the flavo-hydroquinone regenerating the pool of oxidized flavins. The proposed mechanism represents a photocycle in which two antagonistic photoreceptor forms, Fl and FlH₂, determine the pool size of the biological effector molecule, the flavo-semiquinone. The redox changes that are associated with the photocycle are maintained by redox partners, pterins, that function in the near-UV as secondary chromophores.Abbreviations FAD flavin adenine dinucleotide - Fl oxidized flavin - FlH flavo-semiquinone radical - FlH₂ flavo-hydroquinone - LIAC light-induced absorbance change - LIFC light-induced fluorescence change - Pt oxidized pterin - PtH₂ dihydro-pterin - PtH₄ tetrahydro-pterin     

Effect of redox mediators on nitrogenase and hydrogenase activities in Azotobacter vinelandii

In bioelectrochemical studies, redox mediators such as methylene blue, natural red, and thionine are used to studying the redox characteristics of enzymes in the living cell. Here we show that nitrogenase activity in Azotobacter vinelandii is completely inhibited by oxidized methylene blue (MB_o) when the concentration of this mediator in the medium is increased up to 72 M. This activity in A. vinelandii is somewhat inhibited by a coenzyme, ascorbic acid (AA). However, the nitrogenase activity within the A. vinelandii cell is unchanged even for a high concentration of oxidized natural red (NR_o) alone. Interestingly, these mediators and AA do not have the capacity to inhibit the H₂ uptake activity of the hydrogenase in A. vinelandii. Average active rates of 66 nM H₂ evolved/mg cell protein/min from the nitrogenase and 160 nM H₂-uptake/mg cell protein/min from the hydrogenase in A. vinelandii are found in aid of the activities of the enzymes for H₂ evolution and for H₂ uptake are compared. The activities of both enzymes in A. vinelandii are strongly inhibited by thionine having high oxidative potential. Mechanisms of various mediators acting in vivo for both enzymes in A. vinelandii are discussed.     

Adenylate energy charge ofAzotobacter vinelandii during encystment

The energy charge ofAzotobacter vinelandii was measured and found to be poised at a value of 0.3. This value was expected in view of the rate of oxygen consumption observed in these resting cells. Adenine nucleotides and poly-β-hydroxybutyric acid were also determined and the relative amounts of these were used as a basis for speculation on long term survival ofAzotobacter cysts in soil. *** DIRECT SUPPORT *** A01R4012 00003     

Uncoupled redox systems in the lumen of the endoplasmic reticulum. Pyridine nucleotides stay reduced in an oxidative environment

The redox state of the intraluminal pyridine nucleotide pool was investigated in rat liver microsomal vesicles. The vesicles showed cortisone reductase activity in the absence of added reductants, which was dependent on the integrity of the membrane. The intraluminal pyridine nucleotide pool could be oxidized by the addition of cortisone or metyrapone but not of glutathione. On the other hand, intraluminal pyridine nucleotides were slightly reduced by cortisol or glucose 6-phosphate, although glutathione was completely ineffective. Redox state of microsomal protein thiols/disulfides was not altered either by manipulations affecting the redox state of pyridine nucleotides or by the addition of NAD(P)+ or NAD(P)H. The uncoupling of the thiol/disulfide and NAD(P)+/NAD(P)H redox couples was not because of their subcompartmentation, because enzymes responsible for the intraluminal oxidoreduction of pyridine nucleotides were distributed equally in smooth and rough microsomal subfractions. Instead, the phenomenon can be explained by the negligible representation of glutathione reductase in the endoplasmic reticulum lumen. The results demonstrated the separate existence of two redox systems in the endoplasmic reticulum lumen, which explains the contemporary functioning of oxidative folding and of powerful reductive reactions.     

Spectral studies on cytochrome d ofAzotobacter vinelandii

Carbon monoxide binding and photodissociation (photolysis) properties of cytochrome d in the membrane electron transport particles ofAzotobacter vinelandii were investigated at cold temperature. The Soret absorption spectrum of cytochrome d was revealed. The evidence supports the thesis that cytochrome d is the major oxidase that carries out NADH oxidative pathway to oxygen at the late-log stage of cell growth inAzotobacter vinelandii.     

Cofactor engineering in Saccharomyces cerevisiae: Expression of a H2O-forming NADH oxidase and impact on redox metabolism

The pyridine nucleotides NAD(H) and NADP(H) play major roles in the formation of by-products. To analyse how Saccharomyces cerevisiae (S. cerevisiae) metabolism during growth on glucose might be altered when intracellular NADH pool is decreased, we expressed noxE encoding a water-forming NADH oxidase from Lactococcus lactis (L. lactis) in the S. cerevisiae strain V5. During batch fermentation under controlled microaeration conditions, expression of the NADH oxidase under the control of a yeast promoter lead to large decreases in the intracellular NADH concentration (five-fold) and NADH/NAD+ ratio (six-fold). This increased NADH consumption caused a large redistribution of metabolic fluxes. The ethanol, glycerol, succinate and hydroxyglutarate yields were significantly reduced as a result of the lower NADH availability, whereas the formation of more oxidized metabolites, acetaldehyde, acetate and acetoin was favoured. The biomass yield was low and consumption of glucose, at concentration above 10%, was impaired. The metabolic redistribution in cells expressing the NADH oxidase was a consequence of the maintenance of a redox balance and of the management of acetaldehyde formation, which accumulated at toxic levels early in the process.     

Cellular redox state protects acetaldehyde-induced alteration in cardiomyocyte function by modifying Ca2+ release from sarcoplasmic reticulum

Recent studies indicate that low concentrations of acetaldehyde may function as the primary factor in alcoholic cardiomyopathy by disrupting Ca(2+) handling or disturbing cardiac excitation-contraction coupling. By producing reactive oxygen species, acetaldehyde shifts the intracellular redox potential from a reduced state to an oxidized state. We examined whether the redox state modulates acetaldehyde-induced Ca(2+) handling by measuring Ca(2+) transient using a confocal imaging system and single ryanodine receptor type 2 (RyR2) channel activity using the planar lipid bilayer method. Ca(2+) transient was recorded in isolated rat ventricular myocytes with incorporated fluo 3. Intracellular reduced glutathione level was estimated using the monochlorobimane fluorometric method. Acetaldehyde at 1 and 10 microM increased Ca(2+) transient amplitude and its relative area in intact myocytes, but acetaldehyde at 100 microM decreased Ca(2+) transient area significantly. Acetaldehyde showed a minor effect on Ca(2+) transient in myocytes in which intracellular reduced glutathione content had been decreased against challenge of diethylmaleate to a level comparable to that induced by exposure to approximately 50 microM acetaldehyde. Channel activity of the RyR2 with slightly reduced cytoplasmic redox potential from near resting state (-213 mV) or without redox fixation was augmented by all concentrations of acetaldehyde (1-100 microM) used here. However, acetaldehyde failed to activate the RyR2 channel, when the cytoplasmic redox potential was kept with a reduced (-230 mV) or markedly oxidized (-180 mV) state. This result was similar to effects of acetaldehyde on Ca(2+) transient in diethylmaleate-treated myocytes, probably being in oxidized redox potential. The present results suggest that acetaldehyde acts as an RyR2 activator to disturb cardiac muscle function, and redox potential protects the heart from acetaldehyde-induced alterations in myocytes.     

Microbial growth on 2-bromobutane

A member of the genus Arthrobacter was isolated which grew at the expense of 2-bromobutane as sole source of carbon and energy. Evidence is presented which suggests that the initial conversion of 2-bromobutane to 2-butanol is a spontaneous chemical hydrolysis and not mediated by the organism. Further evidence from oxygen consumption experiments indicates that 2-bromobutane is oxidized through 2-butanol, methyl ethyl ketone, ethyl acetate to acetate and ethanol. Results of experiments with cells grown on pathway intermediates reveal that the enzymes necessary for the oxidation of 2-butanol, methyl ethyl ketone, ethyl acetate, ethanol and acetaldehyde are not coordinately, but individually induced by their respective substrates.     

Regulation of respiration and nitrogen fixation in different types of Azotobacter vinelandii.

The levels of the adenine nucleotides, pyridine nucleotides and the kinetical parameters of the enzymes of the Entner-Doudoroff pathway (glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase) were determined in Azotobacter vinelandii cells, grown under O2- or N2-limiting conditions. It was concluced that the levels of both the adenine nucleotides and pyridine nucleotides do not limit the rate of sucrose oxidation. Experiments with radioactive pyruvate and sucrose show that the rate of sucrose oxidation of Azotobacter cells is associated with an increase in the rate of sucrose uptake. The sites of oxidative phosphorylation and the composition of the respiratory membranes with respect to cytochromes c4 + c5, b and d differ in cells growth either O2- or N2-limited. It was possible to show that the respiration protection of the nitrogen-fixing system in Azotobacter is mainly independent of the oxidation capacity of the cells. The oxidation capacity intrinsically depends on the type of substrate and can be partly adapted. The maximum activity of the nitrogenase in Azotobacter depends on the type of substrate oxidized. Although the level of energy charge is somewhat dependent on the type of substrate used, no obvious relation can be derived between changes in energy charge and nitrogenase activity. An alternative proposal is given.     

Hepatic redox state: attenuation of the acute effects of ethanol induced by chronic ethanol consumption

The effects of an acute dose of a diet containing ethanol (3g/kg) on hepatic redox state was compared in rats fed ethanol for 25 days and in their littermates given isocaloric carbohydrate. In both groups, cytoplasmic and mitochondrial redox states of pyridine nucleotides shifted to a more reduced level, but the changes were much less extensive in rats chronically fed ethanol. This metabolic adaption may reflect the oxidation of ethanol by a pathway not involving alcohol dehydrogenase, such as the microsomal ethanol osidizing system, which increases in activity after chronic ethanol ingestion.     

Influence of the metabolism of ethanol on the lactate/pyruvate ratio of rat-liver slices

1. The influence of ethanol on the redox level of the redox pair lactate/pyruvate has been studied in experiments with rat-liver slices. 2. Ethanol had no effect on oxygen consumption but strongly depressed carbon dioxide formation. On the assumption that ethanol is oxidized to acetate in the liver slices, it could be calculated that most of the oxygen that disappeared was consumed in this reaction. 3. Addition of ethanol to the incubation medium increased the lactate/pyruvate ratio and when all the ethanol had been oxidized the redox value decreased to the normal again. Ethanol depressed the pyruvate concentration, whereas the lactate concentration was not much influenced. 4. Acetaldehyde in the concentrations present during ethanol oxidation did not influence the lactate/pyruvate ratio. Higher concentrations, however, increased the redox state. 5. Acetate in the concentrations present during ethanol oxidation in the experiments, and also in higher concentrations, did not influence the lactate/pyruvate ratio. 6. The mechanism by which ethanol influences the lactate/pyruvate ratio is discussed.     

High‐resolution crystal structures reveal a mixture of conformers of the Gly61‐Asp62 peptide bond in an oxidized flavodoxin from Bacillus cereus

Flavodoxins (Flds) are small proteins that shuttle electrons in a range of reactions in microorganisms. Flds contain a redox‐active cofactor, a flavin mononucleotide (FMN), and it is well established that when Flds are reduced by one electron, a peptide bond close to the FMN isoalloxazine ring flips to form a new hydrogen bond with the FMN N5H, stabilizing the one‐electron reduced state. Here, we present high‐resolution crystal structures of Flavodoxin 1 from Bacillus cereus in both the oxidized (ox) and one‐electron reduced (semiquinone, sq) state. We observe a mixture of conformers in the oxidized state; a 50:50 distribution between the established oxidized conformation where the peptide bond is pointing away from the flavin, and a conformation where the peptide bond is pointing toward the flavin, approximating the conformation in the semiquinone state. We use single‐crystal spectroscopy to demonstrate that the mixture of conformers is not caused by radiation damage to the crystal. This is the first time that such a mixture of conformers is reported in a wild‐type Fld. We therefore carried out a survey of published Fld structures, which show that several proteins have a pronounced conformational flexibility of this peptide bond. The degree of flexibility seems to be modulated by the presence, or absence, of stabilizing interactions between the peptide bond carbonyl and its surrounding amino acids. We hypothesize that the degree of conformational flexibility will affect the Fld ox/sq redox potential.     

Intracellular flux analysis applied to the effect of dissolved oxygen on hybridomas

Quantitative estimates of intracellular fluxes and measurements of intracellular concentrations were used to evaluate the effect of dissolved oxygen (DO) concentration on CRL 1606 hybridoma cells in batch culture. The estimates of intracellular fluxes were generated by combining material balances with measurements of extracellular metabolite rates of change. Experiments were performed at DO levels of 60% and 1% air saturation, as well as under oxygen-limited conditions. Cell extracts were analyzed to evaluate the effect of DO on the intracellular concentrations of the glutamate dehydrogenase reactants, as well as the redox state of the pyridine nucleotides in the cytosol and mitochondria. The relationship between cell density and pyridine nucleotide redox state was also investigated. Dissolved oxygen concentration had a significant effect on nitrogen metabolism and the flux through glutamate dehydrogenase was found to reverse at low DO, favoring glutamate formation. The NAD in the cytosol and mitochondria was more reduced under low DO conditions while the cytosolic NAD was more oxidized at low DO. Cytosolic NAD was reduced at higher cell densities while the redox states of cytosolic NADP and mitochondrial NAD did not exhibit significant variation with cell density. These results point to the fundamental role of the intracellular oxidation/reduction state in cell physiology and the possibility of controlling physiological processes through modulation of the dissolved oxygen level or the oxidation/reduction potential of the culture.     

Comparative study of the response ofAzotobacter vinelandii andAcetobacter diazotrophicus to changes in pH

Summary Curves were established for the pH response of respiration on eleven substrates byAzotobacter vinelandii andAcetobacter diazotrophicus. With every substrate the optimal pH forA. diazotrophicus was lower than forA. vinelandii. The optimal hydrogen ion concentration forA. diazotrophicus was 5 fold to 365 fold greater than forA. vinelandii depending upon the substrate. In general,A. diazotrophicus supports respiration over a wider pH range than doesA. vinelandii.Dedicated to the memory of Professor John G. Torrey     

Respiratory Protection of Nitrogenase Activity in Azotobacter vinelandii—Roles of the Terminal Oxidases

Nitrogen fixation by aerobic prokaryotes appears paradoxical: the nitrogen-fixing enzymes—nitrogenases—are notoriously oxygen-labile, yet many bacteria fix nitrogen aerobically. This review summarises the evidence that cytochrome bd, a terminal oxidase unrelated to the mitochondrial and many other bacterial oxidases, plays a crucial role in aerotolerant nitrogen fixation in Azotobacter vinelandii and other bacteria by rapidly consuming oxygen during uncoupled respiration. We review the pertinent properties of this oxidase, particularly its complement of redox centres, the catalytic cycle of oxygen reduction, the affinity of the oxidase for oxygen, and the regulation of cytochrome bd gene expression. The roles of other oxidases and other mechanisms for limiting damage to nitrogenase are assessed.     

An essential histidine residue in the catalytic mechanism of mammalian glutathione reductase

Glutathione reductase from human erythrocytes was inactivated by ethoxyformic anhydride, and > 95% activity was lost by modification of about 1–1.5 histidine residues per flavin (or subunit), as measured by the increased absorbance at 240 nm. Full reactivation was obtained with hydroxylamine. The rate of inactivation increased with pH and an apparent pK = 5.9 was obtained for the protolytic dissociation. The modified enzyme was inactive with NADPH and GSSG as substrates, but almost fully active in catalysis of a transhydrogenase reaction involving pyridine nucleotides. The visible absorption spectrum of oxidized or two-electron-reduced enzyme was not changed, but the flavin fluorescence of oxidized enzyme increased 2-fold after the modification. NADPH or NADP⁺ did not protect the enzyme against inactivation. It is concluded that the modification affects a histidine involved in the second half-reaction of the catalysis, i.e. reduction of GSSG by the dithiol of reduced enzyme. Glutathione reductase from three additional mammalian sources was similarly inactivated, but enzyme from yeast was much less inactivated by the corresponding treatment with ethoxyformic anhydride.     

Enzymatic oxidation of p-nitrophenol.

A Moraxella sp. capable of growth with p-nitrophenol was isolated from activated sludge. Differential centrifugation of crude cell extracts gave a membrane preparation that oxidized p-nitrophenol to hydroquinone and nitrite. Enzymatic activity was dependent on the presence of oxygen and reduced pyridine nucleotides and was stimulated by the addition of flavin adenine dinucleotide. Experiments with ¹⁸O₂ showed that the incoming hydroxyl group was derived from molecular oxygen. The soluble fraction prepared from crude cell extracts oxidized hydroquinone to a product whose spectral properties were identical to those reported for γ-hydroxymuconic semialdehyde.