Polarographische Messung der Nitritreduktion von Chlorella im monochromatischen Licht

Summary In green plant cells nitrite is reduced by two systems, one dependent on photosynthesis and the other upon respiration. Using a polarographic method for continuous measurement of nitrite uptake, the relationship between light driven and respiration linked nitrite reduction of Chlorella cells was studied.Photosynthetic nitrite reduction is characterized by a pronounced increase in the velocity of nitrite uptake upon illumination. After the light is turned off the velocity immediately returns to the preillumination value. Photosynthetic nitrite reduction of Chlorella is separated from respiration linked nitrite reduction by illumination with red light under anaerobic conditions; it is stimulated by CO₂ and is inhibited by DCMU, findings which confirm earlier observations.In white light a special blue light stimulation of nitrite uptake is overlapped by photosynthetic nitrite reduction. In contrast to photosynthetic nitrite reduction this type of light stimulation is characterized by a lag period of about I min from the onset of illumination; it continues about 10 min when the light is turned off. It is separated from photosynthetic nitrite reduction by irradiation of the algae with low intensities of short wavelength light (<500 nm). Blue light stimulation of nitrite uptake of Chlorella is strongly dependent on the developmental stage of the cells. It is observed with young cells (autospores) of synchronized algae only.There is no evidence for any connection between blue light stimulation of nitrite uptake and photosynthesis. From the sensitivity of this process towards anaerobic conditions and antimycin A it is concluded to be a stimulation of respiration linked nitrite reduction.Under conditions of low exogenous nitrite concentration a temporary inhibition of steady state dark nitrite reduction appears immediately after the light is turned off. From several observations it is concluded that the inhibition already exists during the preceding illumination and decreases the rate of total nitrite uptake in the light. This process is suppressed by inhibition of respiration as well as by the inhibitor of photosynthesis, DCMU.If nitrate is the source of nitrogen an excretion of nitrite is found following illumination. The kinetics of this process agree with those observed for the light induced inhibition of steady state dark nitrite reduction immediately after illumination.     

Nitrate uptake and extracellular alkalinization by the green alga Hydrodictyon reticulatum in blue and red light

Nitrate uptake and the medium alkalinization related to it were studied with nets of the coenocytic, giant cell, green alga Hydrodictyon reticulatum. A comparison of red, blue and white light irradiation showed no special control of nitrate uptake and of the corresponding alkalinization of the external medium by light quality, but rather a response as expected for the photosynthetic apparatus. In the dark, nitrate uptake rates amounted to one-fifth of those in saturating white light. This is in contrast to the chlorococcal microalga Monoraphidium braunii, where blue light specifically switched on nitrate uptake-dependent alkalinization and where uptake and reduction of nitrate strongly depended on blue light; the rates in pure red light and in the dark being very low. The stoichiometric ratio between nitrate taken up and extracellular alkalinization was close to 1 (0.86) in air with CO2 but close to 2 (1.84) in N2 for nitrate pre-loaded cells. In the absence of any carbon source, a high proportion of the absorbed and reduced nitrogen is released, most of it as ammonium which causes the excess alkalinization and some as nitrite, which lowers the ratio. Nitrite and ammonium release rates under anaerobic, CO2-free conditions were also independent of red or blue light and continued for several hours when the medium was buffered at pH 6. The data indicate that nitrate uptake, but less its reduction, is regulated differently in vacuolate, coenocytic algae from microalgae. In Hydrodictyon, nitrate uptake and reduction seems to be controlled by energy supply; in various microalgae, in addition, it is controlled specifically by blue light.     

Blue Light, a Positive Switch Signal for Nitrate and Nitrite Uptake by the Green Alga Monoraphidium braunii

Blue light was shown to regulate the utilization of oxidized nitrogen sources by green algae, both by activating nitrate reductase and promoting nitrite reductase biosysnthesis (MA Quiñones, PJ Aparicio [1990] Inorganic Nitrogen in Plants and Microorganisms, Springer-Verlag, Berlin, pp 171-177; MA Quiñones, PJ Aparicio [1990] Photochem Photobiol 51: 681-692). The data reported herein show that, when cells of Monoraphidium braunii at pH 8, containing both active nitrate reductase and nitrite reductase, were sparged with CO₂-free air and irradiated with strong background red light, they took up oxidized nitrogen sources only when PAR comprised blue light. The activation of the transport system(s) of either both nitrate and nitrite was very quick and elicited by low irradiance blue light. In fact, blue light appears to act as a switch signal from the environment, since the uptake of these anions immediately ceased when this radiation was turned off. The requirement of blue light for nitrate uptake was independent of the availability of CO₂ to cells. However, cells under high CO₂ tensions, although they showed an absolute blue light requirement to initially establish the uptake of nitrite, as they gained carbon skeletons to allocate ammonia, gradually increased their nitrite uptake rates in the subsequent red light intervals. Under CO₂-free atmosphere, cells irradiated with strong background red light of 660 nanometers only evolved oxygen when they were additionally irradiated with low irradiance blue light and either nitrate or nitrite was present in the media to provide electron acceptors for the photosynthetic reaction.     

Electron donation between copper containing nitrite reductases and cupredoxins: the nature of protein-protein interaction in complex formation.

In denitrifying organisms with copper containing dissimilatory nitrite reductases, electron donation from a reduced cupredoxin is an essential step in the reduction of nitrite to nitric oxide. Copper nitrite reductases are categorised into two subgroups based on their colour, green and blue, which are found in organisms where the cupredoxins are pseudoazurins and azurins, respectively. In view of this and some in vitro electron donation experiments, it has been suggested that copper nitrite reductases have specific electron donors and that electron transfer takes place in a specific complex of the two proteins. We report results from the first comprehensive electron donation experiments using three copper nitrite reductases, one green and two blue, and five cupredoxins, one pseudoazurin and four azurins. Our data show that pseudoazurin can readily donate electrons to both blue and green copper nitrite reductases. In contrast, all of the azurins react very sluggishly as electron donors to the green nitrite reductase. These results are discussed in terms of surface compatibility of the component proteins, complex formation, overall charges, charge distribution, hydrophobic patches and redox potentials. A docking model for the complexes is proposed.     

Kinetic studies of the copper nitrite reductase from Achromobacter cycloclastes and its interaction with a blue copper protein

Transient state, burst and steady state kinetics of reactions of the blue copper nitrite reductase (NIR) and blue copper protein from Achromobacter cycloclastes are investigated. The two copper-containing species are reacted with each other and where possible with dithionite, ascorbate and nitrite. Both copper proteins are fully reduced by dithionite with both S2O4(2-) and SO2-. species active. NIR is only partially reduced by ascorbate in an unusual biphasic reaction consistent with complete reduction of type-one copper followed by partial reduction of type-two copper. The rate of reduction of the type-one copper is accelerated using phenazine methosulfate as mediator. Nitrite can oxidize dithionite-reduced NIR but cannot reduce oxidized NIR. Rate constants were determined for all observed reactions.     

Light-Mediated Nitrite Accumulation during Denitrification by Pseudomonas sp. Strain JR12

The effect of light on the denitrifying characteristics of a nonphotosynthetic denitrifier, Pseudomonas sp. strain JR12, was examined. Already at low light intensities, nitrite accumulated as a result of light inhibition of nitrite but not of nitrate reduction rates. Exposure of this bacterium to light caused a photooxidation of cytochrome c, an intermediate electron carrier in its respiratory pathway. Photoinhibition of nitrite reduction was reversible, as nitrite reduction rates returned to preillumination levels when light-exposed cells were returned to dark conditions. Antimycin A reversed the inhibitory effect of light on nitrite reduction by preventing a reversed electron flow. Aerobic respiration by this bacterium was not affected by light.     

A blue protein as an inactivating factor for nitrite reductase from Alcaligenes faecalis strain S-6

A blue protein with a molecule weight of 12,000 containing 1 atom of type I Cu2+ was purified and crystallized from a denitrifying bacterium, Alcaligenes faecalis strain S-6, as an inactivating factor for copper-containing nitrite reductase of the same organism. Inactivation of the enzyme occurred when the enzyme was incubated aerobically with a catalytic amount of the blue protein in the presence of reducing agents such as cysteine and ascorbate. The blue protein acts as a direct electron donor for the enzyme to catalyze the reduction of nitrite, but in the absence of nitrite, the enzyme-reduced blue protein system reacts with oxygen to produce H2O2. A suicide inactivation mechanism of the enzyme due to this H2O2 production is proposed.     

Comment of Egami's concept of the evolution of nitrate respiration

Recent results suggest that the presence of common nitrogen salts (sodium nitrite and nitrate) in the irradiation medium can markedly protect filamentous blue green algae from potentially lethal ultraviolet light irradiation. Our results as well as general biological arguments as presented by Egami (above) support and extend Egami's original view that anaerobic respiratory pathways using nitrite and nitrate as terminal electron acceptors evolved prior to oxygen requiring aerobic respiratory pathways.     

Spectroscopic evidence for a copper-nitrosyl intermediate in nitrite reduction by blue copper-containing nitrite reductase

The reactions of nitrogen monoxide (NO) with the blue copper-containing nitrite reductases from Alcaligenes sp. NCIB 11015 and Achromobacter cycloclastes IAM 1013 were investigated spectroscopically. The electron paramagnetic resonance (EPR) signals of the blue coppers vanished in the presence of NO at 77 K, being fully restored by the removal of NO. The additions of NO to the enzyme solutions resulted in the substantial bleaching of the visible absorption bands at room temperature. The reactions were also completely reversible. These results suggest the formation of a cuprous nitrosyl complex (Cu+-NO+), which is likely the intermediate in the enzymatic nitrite reduction.     

Effect of light quality on the organization of photosynthetic electron transport chain of pea seedlings

The activity of NADP and O₂ photoreduction by water is essentially higher in chloroplasts isolated from pea seedlings (Pisum sativum L.) grown under blue light as compared with that from plants grown under red light. In contrast, the photoreduction of NADP and O₂ with photosystem I only is practically the same or even lower in chloroplasts isolated from plants grown under blue light. The addition of plastocyanin does not affect the rate or the extent of NADP photoreduction by water in the chloroplasts isolated from plants grown under blue light, whereas it sharply activates NADP reduction in the chloroplasts isolated from plants grown under red light. The extent of the light-induced oxidation of cytochrome f is appreciably higher in chloroplasts isolated from plants grown under blue light. Cytochrome b₅₅₉ plays the predominant role in the oxidoreductive reactions of these chloroplasts. Furthermore, the fluorescence measurements indicate more effective transfer of excitation energy from chlorophyll to the photosystem II reaction center in chloroplasts isolated from plants grown under blue light.     

Nitrite Reduction in Reconstituted and Whole Spinach Chloroplasts during Carbon Dioxide Reduction

Nitrite reduction in either whole, isolated spinach chloroplasts (Spinacia oleracea L.) or in reconstituted spinach chloroplasts is stimulated by a short period of photosynthetic CO₂ fixation in the light prior to nitrite addition. With reconstituted chloroplasts, a similar stimulation can be obtained in nitrite reduction without CO₂ fixation by the addition of dihydroxyacetone phosphate or fructose 6-phosphate. Specific intermediate metabolites of the photosynthetic carbon reduction cycle may have a regulatory role in nitrite reduction in chloroplasts in the light.     

锰——超氧化物歧化酶活力测定的五种方法比较研究

 本文分别以CN~-抑制和SDS处理区分Mn-SOD与CuZn-SOD,对五种SOD活力测定方法进行了比较研究。结果表明:(1)化学发光法和光化学扩增法不适用于Mn-SOD活力测定,CN~-和SDS对这两种方法有明显的干扰作用。(2)NBT还原、Cyt c还原和亚硝酸盐形成法都能用于Mn-SOD活力测定,用这三种方法测得Mn-SOD每活力单位相当于酶的含量分别为2.93μg、0.11μg和0.028μg。说明NBT还原法灵敏度最低,其次是Cyt c还原法。亚硝酸盐形成法灵敏度高,专一性强,为五种测定方法之首。     

NarK enhances nitrate uptake and nitrite excretion in Escherichia coli.

narK mutants of Escherichia coli produce wild-type levels of nitrate reductase but, unlike the wild-type strain, do not accumulate nitrite when grown anaerobically on a glucose-nitrate medium. Comparison of the rates of nitrate and nitrite metabolism in cultures growing anaerobically on glucose-nitrate medium revealed that a narK mutant reduced nitrate at a rate only slightly slower than that in the NarK+ parental strain. Although the specific activities of nitrate reductase and nitrite reductase were similar in the two strains, the parental strain accumulated nitrite in the medium in almost stoichiometric amounts before it was further reduced, while the narK mutant did not accumulate nitrite in the medium but apparently reduced it as rapidly as it was formed. Under conditions in which nitrite reductase was not produced, the narK mutant excreted the nitrite formed from nitrate into the medium; however, the rate of reduction of nitrate to nitrite was significantly slower than that of the parental strain or that which occurred when nitrite reductase was present. These results demonstrate that E. coli is capable of taking up nitrate and excreting nitrite in the absence of a functional NarK protein; however, in growing cells, a functional NarK promotes a more rapid rate of anaerobic nitrate reduction and the continuous excretion of the nitrite formed. Based on the kinetics of nitrate reduction and of nitrite reduction and excretion in growing cultures and in washed cell suspensions, it is proposed that the narK gene encodes a nitrate/nitrite antiporter which facilitates anaerobic nitrate respiration by coupling the excretion of nitrite to nitrate uptake. The failure of nitrate to suppress the reduction of trimethylamine N-oxide in narK mutants was not due to a change in the level of trimethylamine N-oxide reductase but apparently resulted from a relative decrease in the rate of anaerobic nitrate reduction caused by the loss of the antiporter system.     

The Effect of Inhibitors of Photosynthetic and Respiratory Electron Transport on Nitrate Reduction and Nitrite Accumulation in Excised Z. mays L. Leaves

The assimilation of nitrate and nitrite under dark and lightconditions in Zea mays L. leaves was investigated. Nitrate wasassimilated under dark-aerobic conditions. Anaerobiosis stimulatednitrate reduction and nitrite accumulation under dark conditions.Vacuum infiltration of inhibitors of respiratory electron transport,antimycin A and rotenone, stimulated nitrate reduction and nitriteaccumulation under dark-aerobic conditions. Vacuum infiltrationof low concentrations of PCP, DNP and mCCCP depressed nitratereduction and nitrite accumulation under dark-aerobic conditions,whereas, infiltration of higher concentrations stimulated nitratereduction and nitrite accumulation. The greatest level of nitrateand nitrite reduction occurred under light conditions. The inhibitorof photosynthetic electron transport, DCMU, stimulated the accumulationof nitrite in the light, but decreased nitrate reduction. Whenthe inhibitors of respiratory electron transport antimycin Aand rotenone, were supplied together with DCMU in the light,nitrite accumulation was enhanced. Low concentrations of mCCCPdecreased both nitrate reduction and nitrite accumulation underlight conditions when supplied with DCMU. Key words: Nitrate reduction, Nitrite accumulation, Leaves     

Effects of D-erythrose on nitrogenase activity in whole filaments of Anabaena sp. strain 7120.

D-Erythrose, which has been shown to enhance nitrogenase activity (acetylene reduction) by isolated heterocysts, was studied for its effects on nitrogenase activity and nitrite uptake by whole filaments of Anabaena sp. strain 7120. D-Erythrose had little effect on acetylene reduction in the light; however, at a concentration of 10 mM, it could restore 3'-(3,4-dichlorophenyl)-1',1'-dimethyl urea-inhibited or dark-limited levels to light-supported levels. Sucrose, glucose, or fructose did not exhibit similar effects. D-Erythrose had little effect on nitrite uptake, an indirect measure of nitrite reductase activity by nitrate-grown whole filaments. It was concluded that erythrose effects were mediated by heterocysts and were therefore specific for nitrogenase.     

Molecular biology, genetics and regulation of nitrite reduction in higher plants

Nitrite reductase (ferredoxin:nitrite oxidoreductase, EC 1.6.6.1) carries out the six-electron reduction of nitrite to ammonium ions in the chloroplasts/plastids of higher plants. The complete or partial nucleotide sequences of a number of nitrite reductase apoprotein genes or cDNAs have been determined. Deduced amino acid sequence comparisons have identified conserved regions, one of which probably is involved in binding the sirohaem/4Fe4S centre and another in binding the electron donor, reduced ferredoxin. The nitrite reductase apoprotein is encoded by the nuclear DNA and is synthesised as a precursor carrying an N-terminal extension, the transit peptide, which acts to target the protein to, and within, the chloroplast/plastid. In those plants examined the number of nitrite reductase apoprotein genes per haploid genome ranges from one (barley, spinach) to four ( Nicotiana tabacum ). Mutants defective in the nitrite reductase apoprotein gene have been isolated in barley. During plastidogenesis in etiolated plants, synthesis of nitrite reductase is regulated by nitrate, light (phytochrome), and an uncharacterised 'plastidic factor' produced by functional chloroplasts. In leaves of green, white-light-grown plants up-regulation of nitrite reductase synthesis is achieved via nitrate and light and down-regulation by a nitrogenous end-product of nitrate assimilation, perhaps glutamine. A role for phytochrome has not been demonstrated in green, light-grown plants. Light regulation of nitrite reductase genes is related more closely to that of photosynthetic genes than to the nitrate reductase gene. In roots of green, white-light-grown plants nitrate alone is able to bring about synthesis of nitrite reductase, suggesting that the root may possess a mechanism that compensates for the light requirement seen in the leaf.     

The role of light in nitrite reduction; Studies with leaf discs

Summary The reduction of nitrite by leaf discs has been studied. In short term experiments the reduction is markedly stimulated by light, but is not affected by the absence of oxygen or carbon dioxide from the gas phase. Carbon dioxide assimilation is more sensitive than nitrite reduction to 3-(3,4-dichloro-)-1,1-dimethyl urea (DCMU) inhibition. Uncouplers such as carbonyl cyanide m-chlorophenyl-hydrazone (CCCP) do not inhibit nitrite reduction although dinitrophenol (DNP) has a small effect.Although nitrite stimulates oxygen evolution in the light in the absence of CO₂ the stoichiometry of nitrite reduction to oxygen evolution is much less than would be predicted if nitrite is simply acting as a classical Hill reagent.Abbreviations DCMU 3-(3,4-dichlorophenyl)-1,1-dimethyl urea - DNP 2,4-dinitrophenol - CCCP carbonyl cyanide m-chlorophenylhydrazone     

Redox Processes in the Blue Light Response of Guard Cell Protoplasts of Commelina communis L

Guard cell protoplasts from Commelina communis L. illuminated with red light responded to a blue light pulse by an H⁺ extrusion which lasted for about 10 minutes. This proton extrusion was accompanied by an O₂ uptake with a 4H⁺ to O₂ ratio. The response to blue light was nil in darkness without a preillumination period of red light and increased with the duration of the red light illumination until about 40 minutes. However, acidification in response to a pulse of blue light was obtained in darkness when external NADH (1 millimolar) was added to the incubation medium, suggesting that redox equivalents necessary for the expression of the response to blue light in darkness may be supplied via red light. In accordance with this hypothesis, the photosystem II inhibitor 3-(3,4-dichlorophenyl)-1, 1-dimethylurea (10 micromolar) decreased the acidification in response to blue light more efficiently when it was added before red light illumination than before the blue light pulse. In the presence of hexacyanoferrate, the acidification in response to a blue light pulse was partly inhibited (53% of control), suggesting a competition for reducing power between ferricyanide reduction and the response to blue light.     

Light and dark assimilation of nitrate in plants

Abstract. Heterotrophic assimilation of nitrate in roots and leaves in darkness is closely linked with the oxidative pentose phosphate pathway. The supply of glucose-6-phosphate to roots and chloroplasts in leaves in darkness is essential for assimilation of nitrite into amino acids. When green leaves are exposed to light, the key enzyme, glucoses-phosphate dehydrogenase, is inhibited by reduction with thioredoxin. Hence the dark nitrate assimilatory pathway is inhibited under photoautotrophic conditions and replaced by regulatory reactions functioning in light. On account of direct photo-synthetic reduction of nitrite in chloroplasts and availability of excess NADH for nitrate reduclase, the rate of nitrate assimilation is extremely rapid in light. Under dark anaerobic conditions also nitrate is equally rapidly reduced to nitrite on account of abolition of competition for NADH between nitrate reductase and mitochondrial oxidation.     

Occurrence of nitrite reductase in citrus leaves

Evidence is presented for the presence of nitrite reductasein citrus leaves. The enzyme has a Km for nitrite of 45 mu andis inhibited by cyanide. However, unlike citrus nitrate reductase(l), it is probably not a metalloflavo protein, although itmay be related to iron. In addition to the enzymatic nitrite reduction, non-enzymaticnitrite reduction was present in citrus leaf preparations. Underin vivo assay conditions nitrite reduction in one-month-oldleaves was not inhibited by cyanide, in contrast with three-month-oldleaves in which nitrite reduction was almost completely inhibited.Thus it appears that in very young citrus leaves most of thenitrite reduction is non-enzymatic. ¹ Contribution from The Volcani Center, Agricultural ResearchOrganization, P. O. B. 6, Bet Dagan, Israel. Series 1972.........2256AE. (Received November 28, 1972; )