Comparison of carbon monoxide,nitric oxide,and nitrite as inhibitors of the nitrogenase from Clostridium pasteurianum

A comparative study of CO, NO, and nitrite as inhibitors of nitrogenase has been carried out. Confirming previous studies, we found that CO inhibits acetylene reduction, but not H₂ evolution nor ATP hydrolysis. On the other hand, NO and nitrite both inhibit acetylene reduction, H₂ evolution, and ATP hydrolysis. Nitrogenase inhibition by CO is readily reversible, whereas the effects of NO and nitrite are irreversible. NO was found to inactivate rapidly and irreversibly the Fe protein, but not the Mo-Fe protein. In the presence of NO, part of the iron of the Fe protein is complexed by bathophenanthrolinedisulfonate, which suggests that NO disrupts the Fe₄S₄ cluster present in the protein. Like NO, nitrite reacts preferentially with the Fe protein, and it also induces complexation of the iron by bathophenanthrolinedisulfonate. We found that under the conditions normally used for the assay of nitrogenase, nitrite is reduced by dithionite. Even though the latter reaction proceeds at a very low rate, enough NO is evolved to inhibit nitrogenase. In view of the striking similarities between the inhibitory effects of NO and nitrite, we suggest that nitrogenase may be inhibited not by nitrite itself, but rather by the nitric oxide produced by the reduction of nitrite.     

Inhibition of electron transfer through the cytochrome b-c 1 complex by nitric oxide in a photodenitrifier,Rhodopseudomonas sphaeroides forma sp. denitrificans

The effects of nitric oxide (NO) on electron transfer were studied with a photodenitrifier, Rhodopseudomonas sphaeroides forma sp. denitrificans. NO inhibited the oxidation of cytochrome c induced by continuous illumination in intact cells. NO inhibited the re-reduction of cytochrome c, the slow phase of the carotenoid bandshift, and the oxidation of cytochrome b after a flash illumination, suggesting that NO inhibited the photosynthetic cyclic electron transfer through the cytochrome b-c ₁ region. NO also inhibited the nitrite (NO ₂ ^- ) and NO reductions with succinate as the electron donor in intact cells, but did not inhibit the NO ₂ ^- and NO reductions in chromatophore membranes with ascorbate and phenazine methosulfate as the electron donors. NO reversibly inhibited the ubiquinol: cytochrome c oxidoreductase of the membranes, suggesting that NO inhibited the electron transfer through the cytochrome b-c ₁ region and that the cytochrome b-c ₁ complex also was involved in the electron transport in both NO ₂ ^- and NO reductions. The catalytic site of NO reduction was distinct from the inhibitory site of NO.Abbreviations UHDBT 5-undecyl-6-hydroxy-4,7-dioxobenzothiazole - UHNQ 3-undecyl-2-hydroxy-1,4-naphthoquinone - MOPS 3-(N-morpholino)propane-sulfonic acid - PMS phenazine methosulfate - DCIP 2,6-dichlorophenol indophenol - DDC diethyl-dithiocarbamate     

Nitrogenase activity and nitrate respiration inAzospirillum spp.

The interaction between nitrate respiration and nitrogen fixation inAzospirillum lipoferum andA. brasilense was studied. All strains examined were capable of nitrogen fixation (acetylene reduction) under conditions of severe oxygen limitation in the presence of nitrate. A lag phase of about 1 h was observed for both nitrate reduction and nitrogenase activity corresponding to the period of induction of the dissimilatory nitrate reductase. Nitrogenase activity ceased when nitrate was exhausted suggesting that the reduction of nitrate to nitrite, rather than denitrification (the further reduction of nitrite to gas) is coupled to nitrogen fixation. The addition of nitrate to nitrate reductase negative mutants (nr^-) ofAzospirillum did not stimulate nitrogenase activity. Under oxygen-limited conditionsA. brasilense andA. lipoferum were also shown to reduce nitrate to ammonia, which accumulated in the medium. Both species, including strains ofA. brasilense which do not possess a dissimilatory nitrite reductase (nir^-) were also capable of reducing nitrous oxide to N₂.     

Nitrate and nitrite reductase negative mutants of N2-fixingAzospirillum spp.

Chlorate resistant spontaneous mutants ofAzospirillum spp. (syn.Spirillum lipoferum) were selected in oxygen limited, deep agar tubes with chlorate. Among 20 mutants fromA. brasilense and 13 fromA. lipoferum all retained their functional nitrogenase and 11 from each species were nitrate reductase negative (nr^–). Most of the mutants were also nitrite reductase negative (nir^–), only 3 remaining nir⁺. Two mutants from nr⁺ nir⁺ parent strains lost only nir and became like the nr⁺ nir^– parent strain ofA. brasilense. No parent strain or nr⁺ mutant showed any nitrogenase activity with 10 mM NO ₃ ^– . In all nr^– mutants, nitrogenase was unaffected by 10 mM NO ₃ ^– . Nitrite inhibited nitrogenase activity of all parent strains and mutants including those which were nir^–. It seems therefore, that inhibition of nitrogenase by nitrate is dependent on nitrate reduction. Under aerobic conditions, where nitrogenase activity is inhibited by oxygen, nitrate could be used as sole nitrogen source for growth of the parent strains and one mutant (nr^– nir^–) and nitritite of the parent strains and 10 mutants (all types). This indicates the loss of both assimilatory and dissimilatory nitrate reduction but only dissimilatory nitrite reduction in the mutants selected with chlorate.     

Nitrogenase activity and dark CO2 fixation in the lichen Peltigera aphthosa Willd

The lichen Peltigera aphthosa consists of a fungus and green alga (Coccomyxa) in the main thallus and of a Nostoc located in superficial packets, intermixed with fungus, called cephalodia. Dark nitrogenase activity (acetylene reduction) of lichen discs (of alga, fungus and Nostoc) and of excised cephalodia was sustained at higher rates and for longer than was the dark nitrogenase activity of the isolated Nostoc growing exponentially. Dark nitrogenase activity of the symbiotic Nostoc was supported by the catabolism of polyglucose accumulated in the ligh and which in darkness served to supply ATP and reductant. The decrease in glucose content of the cephalodia paralleled the decline in dark nitrogenase activity in the presence of CO₂; in the absence of CO₂ dark nitrogenase activity declined faster although the rate of glucose loss was similar in the presence and absence of CO₂. Dark CO₂ fixation, which after 30 min in darkness represented 17 and 20% of the light rates of discs and cephalodia, respectively, also facilitated dark nitrogenase activity. The isolated Nostoc, the Coccomyxa and the excised fungus all fixed CO₂ in the dark; in the lichen most dark CO₂ fixation was probably due to the fungus. Kinetic studies using discs or cephalodia showed highest initial incorporation of ¹⁴CO₂ in the dark in to oxaloacetate, aspartate, malate and fumarate; incorporation in to alanine and citrulline was low; incorporation in to sugar phosphates, phosphoglyceric acid and sugar alcohols was not significant. Substantial activities of the enzymes phosphoenolpyruvate (PEP) carboxylase (EC 4.1.1.31) and carbamoyl-phosphate synthase (EC 2.7.2.5 and 2.7.2.9) were detected but the activities of PEP carboxykinase (EC 4.1.1.49) and PEP carboxyphosphotransferase (EC 4.1.1.38) were negligible. In the dark nitrogenase activity by the cephalodia, but not by the free-living Nostoc, declined more rapidly in the absence than in the presence of CO₂ in the gas phase. Exogenous NH ₄ ⁺ inhibited nitrogenase activity by cephalodia in the dark especially in the absence of CO₂ but had no effect in the light. The overall data suggest that in the lichen dark CO₂ fixation by the fungus may provide carbon skeletons which accept NH ₄ ⁺ released by the cyanobacterium and that in the absence of CO₂, NH ₄ ⁺ directly, or indirectly via a mechanism which involves glutamine synthetase, inhibits nitrogenase activity.Abbreviations CP carbamoyl phosphate - EDTA ethylenedi-amine tetraacetic acid - PEP phosphoenolpyruvate - RuBP ribulose 1,5 bisphosphate     

Nitrite reduction and superoxide-dependent nitric oxide degradation by Arabidopsis mitochondria: Influence of external NAD(P)H dehydrogenases and alternative oxidase in the control of nitric oxide levels

Mitochondria recently have emerged as important sites in controlling NO levels within the cell. In this study, the synthesis of nitric oxide (NO) from nitrite and its degradation by mitochondria isolated from Arabidopsis thaliana were examined. Oxygen and NO concentrations in the reaction medium were measured with specific electrodes. Nitrite inhibited the respiration of isolated A. thaliana mitochondria, in competition with oxygen, an effect that was abolished or potentiated when electron flow occurred via alternative oxidase (AOX) or cytochrome c oxidase (COX), respectively. The production of NO from nitrite was detected electrochemically only under anaerobiosis because of a superoxide-dependent process of NO degradation. Electron leakage from external NAD(P)H dehydrogenases contributed the most to NO degradation as higher rates of Amplex Red-detected H₂O₂ production and NO consumption were observed in NAD(P)H-energized mitochondria. Conversely, the NO-insensitive AOX diminished electron leakage from the respiratory chain, allowing the increase of NO half-life without interrupting oxygen consumption. These results show that the accumulation of nitric oxide derived from nitrite reduction and the superoxide-dependent mechanism of NO degradation in isolated A. thaliana mitochondria are influenced by the external NAD(P)H dehydrogenases and AOX, revealing a role for these alternative proteins of the mitochondrial respiratory chain in the control of NO levels in plant cells.     

Denitrification by Azospirillum brasilense Sp 7

Nitrous oxide reduction can consistently be demonstrated with high activities in cells of Azospirillum brasilense Sp 7 which are grown anaerobically in the presence of low amounts of nitrite. Azospirillum can even grow anaerobically with nitrous oxide in the absence of any other respiratory electron acceptor. Nitrous oxide reduction by Azospirillum is inhibited by acetylene, amytal and weakly by carbon monoxide. Azospirillum converts nitrous oxide to molecular nitrogen without the formation of ammonia. The cells must, therefore, be supplied with ammonia from nitrogen fixation during anaerobic growth with nitrous oxide. When no other nitrogen compound besides nitrous oxide is available in the medium, the bacteria synthesize nitrogenase from protein reserves in about 2 h. Nitrogenase synthesis is blocked by chloramphenicol under these conditions. In contrast, the addition of nitrate or nitrite to the medium represses the synthesis of nitrogenase. Nitrous oxide reduction by Azospirillum and other microorganisms is possibly of ecological significance, because the reaction performed by the bacteria may remove nitrous oxide from soils.     

The source of the iron binding with nitric oxide in activated macrophages]

No decrease in iron-sulphur centers was found in cultured macrophage cells (J774) after the treatment with nitric oxide (10(-7) M NO/10(7) cells) during 5 min. The center content was controlled by the electron spin resonance (ESR) method. The macrophages pretreated with dithionite + methyl viologen showed the formation of dinitrosyl iron complexes (DNIC) with a characteristic ESR signal at g approximately 2.03. The data suggest that loosely bound nonheme iron (free iron) mostly contributes to the formation of these complexes. Iron from iron-containing proteins does not release from these centers under the direct action of nitric oxide. The iron-sulphur centers can be destroyed by the products of nitric oxide oxidation (NO2, N2O3, etc.) as oxidizing and acid agents.     

Identification of a new class of nitrogen fixation genes in Rhodobacter capsalatus: a putative membrane complex involved in electron transport to nitrogenase

DNA sequence analysis of a 12236 by fragment, which is located upstream of nifE in Rhodobacter capsulatus nif region A, revealed the presence of ten open reading frames. With the exception of fdxC and fdxN, which encode a plant-type and a bacterial-type ferredoxin, the deduced products of these coding regions exhibited no significant homology to known proteins. Analysis of defined insertion and deletion mutants demonstrated that six of these genes were required for nitrogen fixation. Therefore, we propose to call these genes rnfA, rnfB, rnfC, rnfD, rnfE and rnfF (for Rhodobacter nitrogen fixation). Secondary structure predictions suggested that the rnf genes encode four potential membrane proteins and two putative iron-sulphur proteins, which contain cysteine motifs (C-X₂-C-X₂-C-X₃-C-P) typical for [4Fe-4S] proteins. Comparison of the in vivo and in vitro nitrogenase activities of fdxN and rnf mutants suggested that the products encoded by these genes are involved in electron transport to nitrogenase. In addition, these mutants were shown to contain significantly reduced amounts of nitrogenase. The hypothesis that this new class of nitrogen fixation genes encodes components of an electron transfer system to nitrogenase was corroborated by analysing the effect of metronidazole. Both the fdxN and rnf mutants had higher growth yields in the presence of metronidazole than the wild type, suggesting that these mutants contained lower amounts of reduced ferredoxins.     

The impact of organic matter on nitric oxide formation by Nitrosomonas europaea

Chemolithoautotrophically growing cells of Nitrosomonas europaea quantitatively oxidized ammonia to nitrite under aerobic conditions with no loss of inorganic nitrogen. Significant inorganic nitrogen losses occurred when cells were growing mixotrophically with ammonium, pyruvate, yeast extract and peptone. Under oxygen limitation the nitrogen losses were even higher. In the absence of oxygen pyruvate was metabolized slowly while nitrite was consumed concomitantly. Nitrogen losses were due to the production of nitric oxide and nitrous oxide. In mixed cultures of Nitrosomonas and Nitrobacter, strong inhibition of nitrite oxidation was reproducibly measured. NO and ammonium were not inhibitory to Nitrobacter. First evidence is given that hydroxylamine, the intermediate of the Nitrosomonas monooxygenase-reaction, is formed. 0.2 to 1.7 M NH₂OH were produced by mixotrophically growing cells of Nitrosomonas and Nitrosovibrio. Hydroxylamine was both a selective inhibitory agent to Nitrobacter cells and a strong reductant which reduced nitrite to NO and N₂O. It is discussed whether chemodenitrification or denitrification is the most abundant process for NO and N₂O production of Nitrosomonas.     

Nitrite inhibition of nitrogenase from soybean bacteroids

Nitrogenase from soybean bacteroids was purified and used to study NO ₂ ^– effects either as unfractionated enzyme or as reconstituted enzyme from separated nitrogenase components I and II. Partially purified enzyme was strongly inhibited by nitrite at concentrations less than 0.1 mM. This inhibition was typically referred to as competitive with an inhibition constant (K _i) for NO ₂ ^– which was 5.2 mM. Kinetics studies showed an abnormally low apparent constant of association between enzyme and NO ₂ ^– (k _a=60 M^-1·s^-1). Nitrite appeared to bind to the MoFe protein, without any effect on Fe component, giving a completely reversible inhibition. Nitrite was found not to be an alternative substrate for nitrogenase.Abbreviations TES N-tris (hydroxymethyl) methyl-2-aminoethane sulfonic acid - PPG Polypropylene glycol     

Effects of inorganic nitrogen compounds on the activity and synthesis of nitrogenase in Gloeothece (Nägeli) sp. ATCC 27152

Addition of 2 mM nitrite or ammonium to aerobically incubated cultures of Gloeothece rapidly inhibited N₂ fixation (measured as acetylene reduction). In contrast, 2 mM nitrate inhibited N₂ fixation less rapidly and less extensively, and often temporarily stimulated nitrogenase activity. The inhibitory effects of both nitrate and ammonium could be prevented by addition of 3 mM L-methionine-DL-sulphoximine, suggesting that the true inhibitor of N₂ fixation was an assimilatory product of ammonium rather than either ammonium or nitrate itself. The inhibition of N₂ fixation by nitrite could not, however, be prevented by addition of L-methionine-DL- sulphoximine. On the other hand, nitrite (unlike nitrate and ammonium) did not inhibit N₂ fixation in cultures incubated under a gas phase lacking oxygen. These findings suggest that the mechanism whereby nitrite inhibits N₂ fixation in Gloeothece differs from that of either nitrate or ammonium. The inhibitory effect of nitrite on N₂ fixation did not involve reduction of nitrite to nitric oxide, though nitric oxide was a potent inhibitor of nitrogenase activity in Gloeothece . Nitrate and nitrite inhibited the synthesis of nitrogenase in Gloeothece , while ammonium not only inhibited nitrogenase synthesis but also stimulated degradation of the enzyme. In addition, all three compounds favoured the appearance of the Fe-protein of nitrogenase in its larger, presumed inactive, form.     

Denitrification of nitrate to nitrogen gas by washed cells ofRhizobium japonicum and by bacteroids fromGlycine max

Nitrate, nitrite and nitrous oxide were denitrified to N₂ gas by washed cells ofRhizobium japonicum CC706 as well as by bacteroids prepared from root nodules ofGlycine max (L.) Merr. (CV. Clark 63). Radiolabelled N₂ was produced from either K¹⁵NO₃ or Na¹⁵NO₂ by washed cells ofRh. japonicum CC705 grown with either nitrate only (5 mM) or nitrate (5 mM) plus glutamate (10 mM). Nitrogen gas was also produced from N₂O. Similar results were obtained with bacteroids ofG. max. The stoichiometry for the utilization of¹⁵NO ₃ ^- or¹⁵NO ₂ ^- and the produciton of¹⁵N₂ was 2:1 and for N₂O utilization and N₂ production it was 1:1. Some of the¹⁵N₂ gas produced by denitrification of¹⁵NO ₃ ^- in bacteroids was recycled via nitrogenase into cell nitrogen.     

Regulation of nitrogenase activity by light in the azolla-anabaena symbiosis

Nitrogenase activity and the rate of photosynthesis were measured simultaneously in Azolla by a continuous gas flow system. The mode of interaction between light, photosynthesis and nitrogenase activity was analysed.Nitrogenase activity dropped off when either Azolla plants or the cyanobiont Anabaena were transferred from light to dark. This decline was immediate and was independent of length or intensity of the prior light phase. Reillumination restored nitrogenase activity.Nitrogenase activity did not depend on the rate of photosynthesis at light intensities below 10 μE m⁻² s⁻¹. Its activity was saturated at 200 μE m⁻² s⁻¹ while CO₂ fixation was saturated at a light intensity of 850 μE m⁻² s⁻¹. Azolla photosynthetic activity followed the absorption spectrum of chlorophyll a, while nitrogenase activity markedly increased between 690 and 710 nm. Inhibition of photosynthesis by DCMU was accompanied by an increase in nitrogenase activity. These results suggest direct light regulation of nitrogenase activity in Azolla independent of CO₂ fixation, and a possible inhibition of nitrogenase activity by the oxygen produced in photosynthesis.     

Acetylcholine inhibits nitric oxide (NO) synthesis in the gastropod nervous system

Acetylcholine (ACh) is one of the main signals regulating nitric oxide synthase (NOS) expression and nitric oxide (NO) biosynthesis in mammals. However, few comparative studies have been performed on the role of ACh on NOS activity in non-mammalian animals. We have therefore studied the cholinergic control of NOS in the snail Helix pomatia and compared the effects of ACh on NO synthesis in the enteric nervous system of the snail and rat. Analyses by the NADPH-diaphorase reaction, immunocytochemistry, purification with ion-exchange chromatography, Western-blot, and quantitative polymerase chain reaction have revealed the expression of neuronal NOS in the rat intestine and of a 60-kDa subunit of NOS in the enteric nerve plexus of H. pomatia. In H. pomatia, quantification of the NO-derived nitrite ions has established that NO formation is confined to the NOS-containing midintestine. Nitrite production can be elevated by L-arginine but inhibited by N_ω-nitro-L-arginine. In rats, ACh moderately elevates nitrite production, whereas ACh, the nicotinic receptor agonists (nicotine, acetyl thiocholine iodide, metacholine) and the cholinesterase inhibitor eserine reduce enteric nitrite formation in snails. The nicotinic receptor antagonist tubocurarine also provokes nitrite liberation, whereas the muscarinic receptor agonists or antagonists have no significant effect in snails. In the presence of EDTA or tetrodotoxin, ACh fails to inhibit nitrite production. In pharmacological studies, we have found that ACh contracts the midintestinal muscles and, in snails, simultaneously reduces the antagonistic muscle relaxant effect of L-arginine. Our experiments provide the first evidence for an inhibitory regulation of neuronal NO synthesis by ACh in an invertebrate species. This article is dedicated to Dr. Gábor Hollósi on the 50th anniversary of his graduation and being a teacher at the University of Debrecen.     

Oligochitosan induced Brassica napus L. production of NO and H2O2 and their physiological function

NO (nitric oxide) and H₂O₂ (hydrogen peroxide) are important signaling molecule in plants. Brassica napus L. was used to understand oligochitosan inducing production of NO (nitric oxide) and H₂O₂ (hydrogen peroxide) and their physiological function. The result showed that the production of NO and H₂O₂ in epidermal cells of B. napus L. was induced with oligochitosan by fluorescence microscope. And it was proved that there was an interaction between NO and H₂O₂ with L-NAME (N^G-nitro-l-arg-methyl eater), which is an inhibitor of NOS (NO synthase) in mammalian cells that also inhibits plant NO synthesis, and CAT (catalase), which is an important H₂O₂ scavenger, respectively. It was found that NO and H₂O₂ induced by oligochitosan took part in inducing reduction in stomatal aperture and LEA protein gene expression of leaves of B. napus L. All these results showed that oligochitosan have potential activities of improving resistance to water stress.     

Ecophysiological aspects of growth and nitrogen fixation inAzospirillum spp.

The nitrogenase activity ofAzospirillum spp. is efficiently regulated by environmental factors. InA. brasilense andA. lipoferum a rapid switch off of nitrogenase activity occurs after the addition of ammonium chloride. As in photosynthetic bacteria, a covalent modification of nitrogenase reductase (Fe-protein) is involved. InA. amazonense, a non-covalent mechanism causes only a partial inhibition of nitrogenase activity after ammonium chloride is added. In anaerobic conditions, nitrogenase reductase is also switched off by a covalent modification inA. brasilense andA. lipoferum. Short-time exposure ofAzospirillum to increased oxygen levels causes a partially reversible inhibition of nitrogenase activity, but no covalent modification is involved.Azospirillum spp. show variations in their oxygen tolerance. High levels of carotenoids confer a slightly improved oxygen tolerance. Certain amino acids (e. g. glutamate, aspartate, histidine and serine) affect growth and nitrogen fixation differently inAzospirillum spp. Amino acids may influence growth and nitrogen fixation ofAzospirillum in the association with plants.Azospirillum brasilense andA. halopraeferens are the more osmotolerant species. They utilize most amino acids poorly and accumulate glycine betaine, which also occurs in osmotically stressed grasses as a compatible solute to counteract osmotic stress. Nitrogen fixation is stimulated by glycine betaine and choline. Efficient iron acquisition is a prerequisite for competitive and aerotoleran growth and for high nitrogenase activity.Azospirillum halopraeferens andA. amazonense assimilate iron reasonably well, whereas growth of someA. brasilense andA. lipoferum strains is severely inhibited by iron limitation and by competition with foreign microbial iron chelators. However, growth of certain iron-limitedA. brasilense strains is stimulated by the phytosiderophore mugineic acid. Thus, various plant-derived substances may stimulate growth and nitrogen fixation ofAzospirillum.     

Glyoxylate decreases the oxygen sensitivity of nitrogenase activity and photosynthesis in the cyanobacterium Anabaena cylindrica

Raising the pO₂ reduced nitrogenase activity (C₂H₂ reduction) of Anabaena cylindrica for both glyoxylate-treated (5 mM) and untreated cells. The stimulation caused by glyoxylate, however, increased with increases of pO₂ from 2 to 99 kPa. As the pO₂ increased the net CO₂ fixation was lowered (Warburg effect) while the CO₂ compensation point increased. Glyoxylate partly relieved this sensitivity of net photosynthesis to oxygen and reduced the compensation point considerably. The cells used were preincubated in the dark to exhaust photosynthetic pools. A more pronounced reduction in sensitivity of nitrogenase to oxygen for glyoxylate-treated cells was evident when a preincubation in air with reduced pCO₂ (13 l l^-1) was used. This was, however, not evident until after a 10-h incubation in air. Before this point 2 kPa O₂ sustained the highest nitrogenase activity. Addition of 0.5 and 5 mM of HCO ₃ ^- to Anabaena cultures preincubated at low CO₂ levels (29 l l^-1) abolished the stimulatory effect of glyoxylate on the nitrogenase. Thus, the results sustain the suggestion that glyoxylate may act as an inhibitor of photorespiratory activities in cyanobacteria and can be used as a means of increasing their nitrogen and CO₂ fixation capacities.Abbreviation RuBP ribulose 1,5-bisphosphate     

The role of nitrite in nitric oxide homeostasis: A comparative perspective

Nitrite is endogenously produced as an oxidative metabolite of nitric oxide, but it also functions as a NO donor that can be activated by a number of cellular proteins under hypoxic conditions. This article discusses the physiological role of nitrite and nitrite-derived NO in blood flow regulation and cytoprotection from a comparative viewpoint, with focus on mammals and fish. Constitutive nitric oxide synthase activity results in similar plasma nitrite levels in mammals and fish, but nitrite can also be taken up across the gills in freshwater fish, which has implications for nitrite/NO levels and nitrite utilization in hypoxia. The nitrite reductase activity of deoxyhemoglobin is a major mechanism of NO generation from nitrite and may be involved in hypoxic vasodilation. Nitrite is readily transported across the erythrocyte membrane, and the transport is enhanced at low O₂ saturation in some species. Also, nitrite preferentially reacts with deoxyhemoglobin rather than oxyhemoglobin at intermediate O₂ saturations. The hemoglobin nitrite reductase activity depends on heme O₂ affinity and redox potential and shows species differences within mammals and fish. The NO forming capacity is elevated in hypoxia-tolerant species. Nitrite-induced vasodilation is well documented, and many studies support a role of erythrocyte/hemoglobin-derived NO. Vasodilation can, however, also originate from nitrite reduction within the vessel wall, and at present there is no consensus regarding the relative importance of competing mechanisms. Nitrite reduction to NO provides cytoprotection in tissues during ischemia-reperfusion events by inhibiting mitochondrial respiration and limiting reactive oxygen species. It is argued that the study of hypoxia-tolerant lower vertebrates and diving mammals may help evaluate mechanisms and a full understanding of the physiological role of nitrite.     

The regulation of nodulation,nitrogen fixation and ammonium assimilation under a carbohydrate shortage stress in the Discaria trinervis-Frankia symbiosis

N₂-fixation is sensitive to limitation in the availability of newly synthesised carbohydrates for the nodules. We decided to explore the response of the D. trinervis - Frankia symbiosis to a transient decrease in carbohydrate supply to nodules. Feedback inhibition of nodulation as well as nodule growth was not released by a 6-day dark stress in D. trinervis nodulated plants. However, nitrogen fixation and assimilation were affected by the imposed stress. Nitrogenase activity was totally inhibited after 4 days of darkness although high levels of nitrogenase components were still detected at this time. Degradation of FeMo and Fe nitrogenase subunits – both at similar rates – was observed after 6 days of dark stress, revealing the need for inactivation to precede enhancement of protein turnover. Glutamine synthetase (GS), malate dehydrogenase (MDH) and asparagine synthetase (AS) polypeptides were also degraded during the dark stress, although at a lower rate than nitrogenase. ARA and nitrogenase were totally recovered 8 days after resuming normal illumination. It seems that current nitrogenase activity and ammonium assimilation are not, or are only weakly linked with the feedback control of nodulation in D. trinervis. These observations give support to the persistence of an autoregulatory signal in mature nodules that is not sensitive to transient shortages of carbon supply and sustains the inhibition of nodulation in the transient absence of N₂ fixation.