CO2 is the first species formed upon CO oxidation by CO dehydrogenase from Pseudomonas carboxydovorans

The active species of CO₂, i.e. CO₂ or HCO ₃ ^- , formed in the CO dehydrogenase reaction was determined using the pure enzyme from the carboxydotrophic bacterium Pseudomonas carboxydovorans. Employing an assay system similar to that used to test for carbonic anhydrase, data were obtained which are quite compatible with those expected if CO₂ is the first species formed. In addition, carbonic anhydrase activity was not detected in P. carboxydovorans.     

Effects of pyridine nucleotides on the gating of ATP-sensitive potassium channels in insulin-secreting cells

Summary The single-channel current recording technique has been used to study the influences that the pyridine nucleotides NAD, NADH, NADP and NADPH have on the gating of ATP-sensitive K⁺ channels in an insulin-secreting cell line (RINm5F). The effects of the nucleotides were studied at the intracellular surface using either excised inside-out membrane patches or permeabilized cells. All four pyridine nucleotides were found to evoke similar effects. At low concentrations, 100 m and less, each promoted channel opening whereas high concentrations, 500 m and above, evoked channel closure. The degree of K⁺ channel activation by pyridine nucleotides (low conc.) was found to be similar to that evoked by the same concentrations of ADP or GTP, whereas the degree of K⁺ channel inhibition (high conc.) was less marked than that evoked by the same concentrations of ATP, and never resulted in refreshment of K⁺ channels following removal. The effects of NAD, NADH, NADP and NADPH seemed to interact with those of ATP and ADP. In the presence of 1mm ADP and 4mm ATP, 10 to 100 m concentrations of the pyridine nucleotides could not evoke channel opening, whereas concentrations of 500 m and above were found to evoke channel closure. In the presence of 2mm ATP and 0.5mm ADP, however, 10 to 100 m concentrations of the pyridine nucleotides were able to activate K⁺ channels.     

The redox levels and subcellular distribution of pyridine nucleotides in illuminated barley leaf protoplasts studied by rapid fractionation

The redox level and compartmentation of pyridine nucleotides was studied under photorespiratory and non-photorespiratory conditions using rapid fractionation of barley ( Hordeum vulgare L. cv. Gunilla, Svalöv) leaf protoplasts. From comparative measurements of the NADPH/NADP⁺ ratio and the ATP/ADP ratio one acidic and one alkaline extraction medium was chosen which quenched the metabolism very efficiently. The mitochondrial NADH/NAD⁺ was higher under photorespiratory conditions than under non-photorespiratory conditions. Aminoacetonitrile, an inhibitor of the photorespiratory conversion of glycine to serine, lowered the mitochondrial NADH/NAD⁺ ratio. This supports the hypothesis that glycine oxidation is coupled to oxidative phosphorylation to provide ATP to the cytosol. The chloroplastic NADPH/NADP⁺ as well as the NADH/NAD⁺ ratios were quite stable in saturating and limiting CO₂ as well as in the presence of aminoacetonitrile, although the triosephosphate/phosphoglycerate ratios changed. Thus, the redox level in the stroma seems to be tightly regulated.     

Genes encoding ribulosebisphosphate carboxylase and phosphoribulokinase are duplicated in Pseudomonas carboxydovorans and conserved in carboxydotrophic bacteria

Heterologous gene probes derived from cfxLp and cfxPp genes of Alcaligenes eutrophus H16 revealed the presence of structural genes encoding ribulosebisphosphate carboxylase (Rubisco) and phosphoribulokinase (PRK) on the genome of carboxydotrophic bacteria. The two genes were found to be rather conserved. In Pseudomonas carboxydovorans OM5 cfx genes reside on the plasmid pHCG3 and the chromosome as well, indicating that they are duplicated. Also in all plasmidharboring carboxydotrophic bacteria cfxL and cfxP structural genes were found to be plasmid-coded. Our results extend the list of carboxydotrophy structural genes residing on the plasmid pHCG3 and strongly support the idea that the components essential for the chemolithoautotrophic utilization of CO by Pseudomonas carboxydovorans OM5 are plasmid-coded. A cfxL gene probe from Rhodospirillum rubrum did not detectably hybridize with DNA from any of the carboxydotrophic bacteria examined.Abbreviations CODH carbon monoxide dehydrogenase - H₂ase hydrogenase - kb kilobase - PRK phosphoribulokinase - Rubisco ribulosebisphosphate carboxylase - SDS sodium dodecylsulfate     

Formation of pyridine nucleotides under symbiotic and non-symbiotic conditions between soybean nodules and free-living rhizobia

Enzymatic regulation of pyricline nucleotide formation, under symbiotic and non-symbiotic conditions, was analyzed using soybeans (Glycine max L. cv. 'Akisengoku') and rhizobia (Bradyrhizobia japonicum strain A1017), respectively. It was found that levels of pyridine nucleotides in bacteroids in root nodules were different from those in free-living cells of rhizobia. This difference was associated with differences in activities of enzymes involved in the pathway from L-tryptophan to NAD and NADP. That is, these activities were lower in bacteroids than in free-living bacteria and lower in the nodule cytosol than in root extracts. The optimum pH for NAD synthetase in bacteroids, was 9.0. Additionally, the optimum pH for ATP-nicotinamide mononucleotide (NMN) adenyltransferase, final step enzyme in NAD formation, was estimated to be 7.6. In the bacteroid fraction, the K(m) of NAD synthetase (22 microM) was approximately 1/22 of that of ATP-NMN adenyltransferase (482 microM). Vmax values were estimated to be almost in the same order for both NAD synthetase and ATP-NMN adenyltransferase. This is the first report on the formation of pyridine nucleotides originating from L-tryptophan in bacteroids in soybean nodules and free-living bacteria.     

The structural genes encoding CO dehydrogenase subunits (cox L,M and S) in Pseudomonas carboxydovorans OM5 reside on plasmid pHCG3 and are,with the exception of Streptomyces thermoautotrophicus,conserved in carboxydotrophic bacteria

Employing deoxyoligonucleotide probes and Southern hybridizations, we have examined in carboxydotrophic bacteria the localization on the genome of genes encoding the large, medium and small subunits of CO dehydrogenase (coxL, M and S, respectively). In Pseudomonas carboxydovorans OM5 coxL, M and S were identified on the plasmid pHCG3; they were absent on the chromosome. This was evident from positive hybridizations with plasmid DNA of the wild-type strain OM5 and the absence of hybridizations with chromosomal DNA from the plasmid cured mutant strain OM5–12. The genes coxL, M and S were found on plasmids in all other plasmid-containing carboxydotrophic bacteria e.g. Alcaligenes carboxydus, Azomonas B1, Pseudomonas carboxydoflava, Pseudomonas carboxydovorans OM2 and OM4. Cox L, M and S could be identified on the chromosome of the plasmid-free bacteria Arthrobacter 11/x, Bacillus schlegelii, Pseudomonas carboxydohydrogena, and Pseudomonas carboxydovorans OM3. These results essentially confirm and extend former reports that cox genes are rather conserved among carboxydotrophic bacteria of distinct taxonomic position. However, Streptomyces thermoautotrophicus is an noteworthy exception since none of the three cox genes could be detected. This refers to a new type of CO dehydrogenase and is in accord with results indicating that the S. thermoautotrophicus CO dehydrogenase has an unusual electron acceptor specificity and some other properties setting it apart from the classical CO dehydrogenases.Abbreviations CODH carbon monoxide dehydrogenase - H₂ase hydrogenase - kb kilobase - PRK phosphoribulokinase - Rubisco ribulosebisphosphate carboxylase - SDS sodium dodecylsulfate     

Iron transport in Francisella in the absence of a recognizable TonB protein still requires energy generated by the proton motive force

The mechanism of iron transport in Francisella is still a puzzle since none of the sequenced Francisella strains appears to encode a TonB protein, the energy transducer of the proton motive force necessary to act on the bacterial outer membrane siderophore receptor to allow the internalization of iron. In this work we demonstrate using kinetic experiments of radioactive Fe³⁺ utilization, that iron uptake in Francisella novicida, although with no recognizable TonB protein, is indeed dependent on energy generated by the proton motive force. Moreover, mutants of a predicted outer membrane receptor still transport iron and are sensitive to the iron dependent antimicrobial compound streptonigrin. Our studies suggest that alternative pathways to internalize iron might exist in Francisella.     

The oxidation of cytosolic NAD(P)H by external NAD(P)H dehydrogenases in the respiratory chain of plant mitochondria

The respiratory chain of plant mitochondria differs from that in mammalian mitochondria by containing several rotenone-insensitive NAD(P)H dehydrogenases. Two of these are located on the outer, cytosolic surface of the inner membrane. One is specific for NADH, the other for NADPH. Only the latter is inhibited by diphenyleneiodonium (DPI). Both of these enzymes are normally dependent upon Ca²⁺ for activity and this constitutes a potentially important mechanism by which the cell can regulate the oxidation of cytosolic NAD(P)H via the concentration of free Ca²⁺. This and other potential regulatory mechanisms such as the substrate concentration and polyamines are discussed.     

NAD(P)H oscillation in relation to the rhythmic contraction in thePhysarum plasmodium

Summary ThePhysarum plasmodium shows rhythmic contractile activities with a period of a few min. Phases of the oscillation in the plasmodium migrating unindirectionally agreed sideways throughout at the frontal part. So, time course of an intracellular chemical component was determined by analyzing small pieces cut off successively from the frontal part of the large plasmodium. Intracellular NAD(P)H concentration oscillated with the same period as the rhythmic contraction but with a different phase advancing about 1/3 of the period. UV irradiation suppressed the rhythmic contraction without affecting the rhythmic variation of NAD(P)H. Thus, the NAD(P)H oscillator works independently of the rhythmic contractile system, but seems entraining with each other.Abbreviations UV ultraviolet - NADH nicotinamide adenine dinucleotide, reduced form - NADPH nicotinamide adenine dinucleotide phosphate, reduced form - ATP adenosine 5-triphosphate - cAMP cyclic adenosine 3, 5-monophosphate - FMNH₂ flavin mononucleotide, reduced form - TCA tricarboxylic acid - BSA bovine serum albumin - DTT dithiothreitol     

Depletion of casein kinase I leads to a NAD(P)/NAD(P)H balance-dependent metabolic adaptation as determined by NMR spectroscopy-metabolomic profile in Kluyveromyces lactis

Background

In the Crabtree-negative Kluyveromyces lactis yeast the rag8 mutant is one of nineteen complementation groups constituting the fermentative-deficient model equivalent to the Saccharomyces cerevisiae respiratory petite mutants. These mutants display pleiotropic defects in membrane fatty acids and/or cell walls, osmo-sensitivity and the inability to grow under strictly anaerobic conditions (Rag⁻ phenotype). RAG8 is an essential gene coding for the casein kinase I, an evolutionary conserved activity involved in a wide range of cellular processes coordinating morphogenesis and glycolytic flux with glucose/oxygen sensing.

Methods

A metabolomic approach was performed by NMR spectroscopy to investigate how the broad physiological roles of Rag8, taken as a model for all rag mutants, coordinate cellular responses.

Results

Statistical analysis of metabolomic data showed a significant increase in the level of metabolites in reactions directly involved in the reoxidation of the NAD(P)H in rag8 mutant samples with respect to the wild type ones. We also observed an increased de novo synthesis of nicotinamide adenine dinucleotide. On the contrary, the production of metabolites in pathways leading to the reduction of the cofactors was reduced.

Conclusions

The changes in metabolite levels in rag8 showed a metabolic adaptation that is determined by the intracellular NAD(P)⁺/NAD(P)H redox balance state.

General significance

The inadequate glycolytic flux of the mutant leads to a reduced/asymmetric distribution of acetyl-CoA to the different cellular compartments with loss of the fatty acid dynamic respiratory/fermentative adaptive balance response.     

Respiratory control over photosynthetic electron transport in chloroplasts of higher-plant cells: evidence for chlororespiration

Flash-induced primary charge separation, detected as electrochromic absorbance change, the operation of the cytochrome b/f complex and the redox state of the plastoquinone pool were measured in leaves, protoplasts and open-cell preparations of tobacco (Nicotiana tabacum L.), and in isolated intact chloroplasts of peas (Pisum sativum L.). Addition of 0.5–5 mM KCN to these samples resulted in a large increase in the slow electrochromic rise originating from the electrogenic activity of the cytochrome b/f complex. The enhancement was also demonstrated by monitoring the absorbance transients of cytochrome f and b ₆ between 540 and 572 nm. In isolated, intact chloroplasts with an inhibited photosystem (PS) II, low concentrations of dithionite or ascorbate rendered turnover of only 60% of the PSI reaction centers, KCN being required to reactivate the remainder. Silent PSI reaction centers which could be reactivated by KCN were shown to occur in protoplasts both in the absence and presence of a PSII inhibitor. Contrasting spectroscopic data obtained for chloroplasts before and after isolation indicated the existence of a continuous supply of reducing equivalents from the cytosol.Our data indicate that: (i) A respiratory electron-transport pathway involving a cyanide-sensitive component is located in chloroplasts and competes with photosynthetic electron transport for reducing equivalents from the plastoquinone pool. This chlororespiratory pathway appears to be similar to that found in photosynthetic prokaryotes and green algae. (ii) There is an influx of reducing equivalents from the cytosol to the plastoquinone pool. These may be indicative of a complex respiratory control of photosynthetic electron transport in higher-plant cells.Abbreviations and symbols A₅₁₅ flash-induced electrochromic absorbance change at 515 nm - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - PS photosystem - SHAM salicylhydroxamic acid     

The origin of photosystem-I-mediated electron transport stimulation in heat-stressed chloroplasts

Exposure of isolated chloroplasts of pea (Pisum sativum L.) to temperatures above 35° C leads to a stimulation of photosystem-I-mediated electron transport from dichlorophenolindophenol to methyl viologen. The threshold temperature for this stimulation coincides closely with that for heat-induced inhibition of photosystem-II activity in such chloroplasts. This coincidence is explained in terms of a rearrangement of the thylakoid membrane resulting in the exposure of a new set of donor sites for dichlorophenolindophenol within the cytochrome f/b ₆ complex of the electron-transport chain linking the two photosystems.Abbreviations cyt cytochrome - DBMIB 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone - DCPIP (H₂) 2,6-dichlorophenolindophenol - EDAC ethyldimethylaminopropyl-carbodiimide - MV methyl viologen - PSI, II photosystem I, II - PCy plastocyanin - PQ(H₂) plastoquinone     

Electron transport activities of <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> mutants with impaired chloroplastic NAD(P)H dehydrogenase

The activities of electron transport are compared between wild-type Arabidopsis and two Arabidopsis mutants deficient for the chloroplastic NAD(P)H dehydrogenase (NDH) which catalyzes cyclic electron transport around photosystem I. The quantum yield of photosystem II and the degree of non-photochemical quenching of chlorophyll fluorescence were of similar levels in the two NDH-deficient mutants and the wild type under non-stressed standard growth conditions. Stromal over-reduction was induced in Arabidopsis NDH mutants with high light treatment, as is the case in tobacco NDH mutants. However, unlike tobacco mutants, photoinhibition was not observed in the Arabidopsis NDH mutants.     

Cloning of ndhK from soybean chloroplasts using antibodies raised to mitochondrial complex I

A soybean shoot cDNA expression library was screened with polyclonal antibodies raised against red beet complex I and several clones were identified. One clone, consisting of a 1 kb insert, was fully sequenced. The sequence of 1025 bp was found to contain two extended open reading frames and the proteins encoded were identified as the ndhK and ndhJ products of the chloroplast genome. Nuclear, mitochondrial and chloroplast DNA was isolated and probed with a ndhK-specific probe. The chloroplast DNA contained a single copy of the cloned insert. With nuclear DNA, positively hybridising bands of 1.2, 2.7 and 3.2 kb were observed indicating that at least one gene homologous to ndhK of the chloroplast genome, is also present in the nucleus. The mitochondrial DNA did not hybridise with the ndhK probe. Western analysis of thylakoid proteins with the mitochondrial complex I antibodies revealed several bands. It is suggested that soybean contains two copies of the ndhK gene, one, on the plastid genome, coding for a subunit of a chloroplast NAD(P)H dehydrogenase, and the other, in the nucleus, coding for a subunit of mitochondrial complex I.     

Evidence for electron transport across the plasma membrane of Zea mays root cells

Exogenous ferricyanide is reduced by roots of Z. mays. In contrast to oxidation of exogenous electron donors, ferricyanide reduction occurs mostly at the apical 5 mm of the root. Using just this portion of the root, it is shown that the activity is neither a consequence of uptake of ferricyanide followed by excretion of its reduced form, nor of leakage of a reductant. Addition of ferricyanide for 40 s or 5 min results in an apparent oxidation of NADPH but not of NADH; rates of ferricyanide reduction vary together with levels of NADPH but not of NADH in the presence or absence of oxygen. It is concluded that an enzyme which can oxidize cytoplasmic NADPH and transfer the electrons to an external acceptor exists at the cell surface of maize roots. This finding extends the results of others who showed similar redox activity at the surface of Fe-depleted dicotyledonous roots, and indicates that an energy source other than ATP exists at the cell surface of a variety of plants under unstressed conditions.     

Cyclic electron flow around photosystem I via chloroplast NAD(P)H dehydrogenase (NDH) complex performs a significant physiological role during photosynthesis and plant growth at low temperature in rice

The role of NAD(P)H dehydrogenase (NDH)-dependent cyclic electron flow around photosystem I in photosynthetic regulation and plant growth at several temperatures was examined in rice (Oryza sativa) that is defective in CHLORORESPIRATORY REDUCTION 6 (CRR6), which is required for accumulation of sub-complex A of the chloroplast NDH complex (crr6). NdhK was not detected by Western blot analysis in crr6 mutants, resulting in lack of a transient post-illumination increase in chlorophyll fluorescence, and confirming that crr6 mutants lack NDH activity. When plants were grown at 28 or 35°C, all examined photosynthetic parameters, including the CO(2) assimilation rate and the electron transport rate around photosystems I and II, at each growth temperature at light intensities above growth light (i.e. 800 μmol photons m(-2) sec(-1)), were similar between crr6 mutants and control plants. However, when plants were grown at 20°C, all the examined photosynthetic parameters were significantly lower in crr6 mutants than control plants, and this effect on photosynthesis caused a corresponding reduction in plant biomass. The F(v)/F(m) ratio was only slightly lower in crr6 mutants than in control plants after short-term strong light treatment at 20°C. However, after long-term acclimation to the low temperature, impairment of cyclic electron flow suppressed non-photochemical quenching and promoted reduction of the plastoquinone pool in crr6 mutants. Taken together, our experiments show that NDH-dependent cyclic electron flow plays a significant physiological role in rice during photosynthesis and plant growth at low temperature.     

Regulation of electron transport in maize mesophyll chloroplasts: The relationship between chlorophyll a fluorescence quenching and O2 evolution

The relationship between the redox state of the primary electron acceptor of photosystem II (Q_A) and the rate of O₂ evolution in isolated mesophyll chloroplasts from Zea mays L. is examined using pulse-modulated chlorophyll a fluorescence techniques. A linear relationship between photochemical quenching of chlorophyll fluorescence (q_Q) and the rate of O₂ evolution is evident under most conditions with either glycerate 3-phosphate or oxaloacetate as substrates. There appears to be no effect of the transthylakoid pH gradient on the rate of electron transfer from photosystem II into Q_A in these chloroplasts. However, the proportion of electron transport occurring through cyclic-pseudocyclic pathways relative to the non-cyclic pathway appears to be regulated by metabolic demand for ATP. The majority of non-photochemical quenching in these chloroplasts at moderate irradiances appeared to be energy-dependent quenching.Abbreviations and symbols PSII photosystem II - Fm maximum fluorescence obtained on application of a saturating light pulse - Fo basal fluorescence recorded in the absence of actinic light (i.e. all PSII traps are open) - Fv Fm-Fo - q_Q photochemical quenching - q_NP non-photochemical quenching - q_E energy-dependent quenching of chlorophyll fluorescence     

Changes in the mode of electron flow to photosystem I following chilling-induced photoinhibition in a C3 plant, Cucumis sativus L.

This study provides evidence for enhanced electron flow from the stromal compartment of the photosynthetic membranes to P700⁺ via the cytochrome b₆/f complex (Cyt b₆/f) in leaves of Cucumis sativus L. submitted to chilling-induced photoinhibition. The above is deduced from the P700 oxidation–reduction kinetics studied in the absence of linear electron transport from water to NADP⁺, cyclic electron transfer mediated through the Q-cycle of Cyt b₆/f and charge recombination in photosystem I (PSI). The segregation of these pathways for P700⁺ rereduction were achieved by the use of a 50-ms multiple turnover white flash or a strong pulse of white or far-red illumination together with inhibitors. In cucumber leaves, chilling-induced photoinhibition resulted in ∼20% loss of photo-oxidizible P700. The measurement of P700⁺ was greatly limited by the turnover of cyclic processes in the absence of the linear mode of electron transport as electrons were rapidly transferred to the smaller pool of P700⁺. The above is explained by integrating the recent model of the cyclic electron flow in C₃ plants based on the Cyt b₆/f structural data [Joliot and Joliot (2006) Biochim Biophys Acta 1757:362–368] and a photoprotective function elicited by a low NADP⁺/NAD(P)H ratio [Rajagopal et al. (2003) Biochemistry 42:11839–11845]. Over-reduction of the photosynthetic apparatus results in the accumulation of NAD(P)H in vivo to prevent NADP⁺-induced reversible conformational changes in PSI and its extensive damage. As the ferredoxin:NADP reductase is fully reduced under these conditions, even in the absence of PSII electron transport, the reduced ferredoxin generated during illumination binds at the stromal openings in the Cyt b₆/f complex and activates cyclic electron flow. On the other hand, the excess electrons from the NAD(P)H pool are routed via the Ndh complex in a slow process to maintain moderate reduction of the plastoquinone pool and redox poise required for the operation of ferredoxin:plastoquinone reductase mediated cyclic flow.     

Upregulation of bundle sheath electron transport capacity under limiting light in C4 Setaria viridis

C₄ photosynthesis is a biochemical pathway that operates across mesophyll and bundle sheath (BS) cells to increase CO₂ concentration at the site of CO₂ fixation. C₄ plants benefit from high irradiance but their efficiency decreases under shade, causing a loss of productivity in crop canopies. We investigated shade acclimation responses of Setaria viridis, a model monocot of NADP-dependent malic enzyme subtype, focussing on cell-specific electron transport capacity. Plants grown under low light (LL) maintained CO₂ assimilation rates similar to high light plants but had an increased chlorophyll and light-harvesting-protein content, predominantly in BS cells. Photosystem II (PSII) protein abundance, oxygen-evolving activity and the PSII/PSI ratio were enhanced in LL BS cells, indicating a higher capacity for linear electron flow. Abundances of PSI, ATP synthase, Cytochrome b₆f and the chloroplast NAD(P)H dehydrogenase complex, which constitute the BS cyclic electron flow machinery, were also increased in LL plants. A decline in PEP carboxylase activity in mesophyll cells and a consequent shortage of reducing power in BS chloroplasts were associated with a more oxidised plastoquinone pool in LL plants and the formation of PSII – light-harvesting complex II supercomplexes with an increased oxygen evolution rate. Our results suggest that the supramolecular composition of PSII in BS cells is adjusted according to the redox state of the plastoquinone pool. This discovery contributes to the understanding of the acclimation of PSII activity in C₄ plants and will support the development of strategies for crop improvement, including the engineering of C₄ photosynthesis into C₃ plants.