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1.
Isolated heterocysts of Anabaena 7120 evolve H2 in an ATP-dependent nitrogenase-catalyzed process that is inhibited by N2 and C2H2. Heterocysts have an active uptake hydrogenase that only requires an electron acceptor of positive redox potential, e.g., methylene blue, dichlorophenolindophenol or potassium ferricyanide. O2 supplied at low partial pressures is a very effective physiological oxidant for H2 uptake. High concentrations of O2 are inhibitory to H2 uptake. The oxyhydrogen reaction in heterocysts appears to be mediated by a cytochrome-cytochrome oxidase system, and it supports ATP synthesis via oxidative phosphorylation. Attempts to demonstrate acetylene reduction in isolated heterocysts employing H2 as an electron donor were unsuccessful. It is suggested that the uptake hydrogenase functions to conserve reductant that otherwise would be dissipated via nitrogenase-catalyzed H2 evolution. 相似文献
2.
Occurrence and localization of an uptake hydrogenase were examined in three strains of the blue-green alga, Anabaena. In vivo H2 uptake was detected (0.60-1.44 μmoles/[mg of chlorophyll a per hour]) in all three strains when grown with N2 as the sole source of nitrogen. H2 uptake (in vivo and in vitro) was severely suppressed in cultures grown on NH4+ and lacking heterocysts. H2 uptake in cell-free extracts could be readily measured with a methyl viologen-ferricyanide electron acceptor system. Solubilization kinetics during cavitation of aerobically grown Anabaena 7120 indicates that the uptake hydrogenase is localized solely in the heterocyst. When the same organism is grown on N2/CO2, vegetative cells may account for up to 21% of the total hydrogenase activity in the filaments. The results are discussed in terms of a proposed functional relationship between nitrogenase and hydrogenase. 相似文献
3.
The reversible hydrogenase from Anabaena 7120 appeared when O2 was continuously removed from a growing culture. Activity increased further when cells were incubated under argon in the dark or in the light plus 3-(3,4-dichlorophenyl)-1,1-dimethylurea. Hydrogenase existed in an inactive state during periods of O2 evolution. It could be reductively activated by exposure to reduced methyl viologen or by dark, anaerobic incubation. Hydrogenase-containing cells evolved H2 slowly during dark anaerobic incubations, and the rate of H2 evolution was increased by illumination with low intensity light. Light enhancement of H2 evolution was of short duration and was eliminated by the ferredoxin antagonist disalicylidene diaminopropane. Physiological acceptors that supported H2 uptake included NO3−, NO2−, and HSO3−, and light had a slight influence on the rate of H2 uptake with these acceptors. Low levels of O2 supported H2 uptake, but higher concentrations of O2 inactivated the hydrogenase. Hydrogen uptake with HCO3− as acceptor was the most rapid reaction measured, and it was strictly light-dependent. It occurred only at low light intensities, and higher light intensities restored normal O2-evolving photosynthesis. It is suggested that hydrogenase is present to capture exogenous H2 as a source of reducing equivalents during growth in anaerobic environments. 相似文献
4.
A method is described for the isolation of heterocysts that are virtually free of contaminating cell debris after sonication of aerobically grown Anabaena 7120. Isolated heterocysts reduced acetylene in a light-dependent process in the absence of exogenously provided ATP; heterocysts supplied with ATP and Na2S2O4 reduced acetylene slowly in the dark but still showed a marked light activation. Nitrogenase activity was greatest in fractions containing intact heterocysts. Up to 13% of the activity of the intact filaments was accounted for in the isolated heterocyst preparation.Isolated heterocysts took up O2 in a light-independent process; O2 uptake with added NADP+ was enhanced by pyruvate, isocitrate and intermediates of the oxidative pentose pathway. 相似文献
5.
Hydrogen-supported nitrogenase activity was demonstrated in Anabaena cylindrica cultures limited for reductant. Nitrogen-fixing Anabaena cylindrica cultures sparged in the light with anaerobic gases in the presence of the photosynthesis inhibitor DCMU slowly lost their ability to reduce acetylene in the light under argon but exhibited near normal activities in the presence of 11% H2 (balance argon). The hydrogen-supported nitrogenase activity was half-saturated between 2 and 3% H2 and was strongly inhibited by oxygen (50% inhibition at about 5–6% O2). Batch cultures of Anabaena cylindrica approaching stationary growth phase (“old” cultures) lost nitrogenase-dependent hydrogen evolution almost completely. In these old cultures hydrogen relieved the inhibitory effects of DCMU and O2 on acetylene reduction. Our results suggest that heterocysts contain an uptake hydrogenase which supplies an electron transport chain to nitrogenase but which couples only poorly with the respiratory chain in heterocysts and does not function in CO2 fixation by vegetative cells. 相似文献
6.
Günter A. Peschek 《BBA》1979,548(2):203-215
1. The oxyhydrogen reaction of Anacystis nidulans was studied manometrically and polarographically in whole cells and in cell-free preparations; the activity was found to be associated with the particulate fraction.2. Besides O2, the isolated membranes reduced artificial electron acceptors of positive redox potential; the reactions were unaffected by O2 levels <10–15%; aerobically the artificial acceptors were reduced simultaneously with O2.3. H2-supported O2 uptake was inhibited by CO, KCN and 2-n-heptyl-8-hydroxyquinoline-N-oxide. Inhibition by CO was partly reversed by strong light. Uncouplers stimulated the oxyhydrogen reaction.4. The kinetic properties of O2 uptake by isolated membranes were the same in presence of H2 and of other respiratory substrates.5. Low rates of H2 evolution by the membrane preparations were found in presence of dithionite; methyl viologen stimulated the reaction.6. The results indicate that under certain growth conditions Anacystis synthesizes a membrane-bound hydrogenase which appears to be involved in phosphorylative electron flow from H2 to O2 through the respiratory chain. 相似文献
7.
Molecular hydrogen inhibits nitrogenase activity in Anabaena pre-illuminated with red or blue light. The inhibitory effect of molecular hydrogen decreased in the presence of oxygen and several electron acceptors. When NH4Cl and urea were added simultaneously with molecular hydrogen, marked synergistic inhibitory effects took place. The inhibitory effect of molecular hydrogen disappeared or was weakened after the suppression of hydrogenase activity. The addition of O2 and electron acceptors to systems showed no enhancing effect on the C2H2-reducing activity. 相似文献
8.
Ecological rationale for H2 metabolism during aquatic blooms of the cyanobacterium Anabaena 总被引:1,自引:0,他引:1
Hans W. Paerl 《Oecologia》1980,47(1):43-45
Summary Nitrogenase-produced H2 serves to remove excess intracellular O2 during vigorous growth periods (blooms) of the nuisance cyanobacterium Anabaena. In two naturally-occurring species, A. oscillarioides and A. spiroides, nitrogen fixation (acetylene reduction) showed a high degree of resistance to O2 inactivation. Under the influence of supersaturated O2 concentrations, commonly encountered in lake blooms, elevated cellular ATP levels and enhanced uptake hydrogenase and nitrogenase activities were observed in actively growing filaments. Oxygen enhancement of nitrogenase activity appears mediated through localized uptake hydrogenase reactions. Hydrogen assimilated by hydrogenase is combined with O2 in a Knallgas reaction, leading to the formation of H2O and ATP via a respiratory chain. This combination of activities appears poised at O2 removal and allows Anabaena to dominate O2 supersaturated surface waters while maintaining optimal nitrogenase activity. Hence, instead of being a wasteful dissipation of reducing power, H2 evolution via nitrogenase ultimately affords protection from O2 while constituting a source of ATP through subsequent H2 metabolism. 相似文献
9.
The role of the oxyhydrogen reaction in the nitrogen metabolism of Anabaena cylin-drica, particularly under conditions of dinitrogen starvation, was investigated. It was shown that although this reaction supports nitrogenase activity in the dark, when the cells are deprived of nitrogen the rate of hydrogen uptake is little changed. Measurements of ammonia excretion into the medium in the presence of methionine sulfoximine under such conditions indicated that hydrogen uptake supported the turnover of cell protein as an alternative source of nitrogen. In the absence of H2 and O2 in the dark, nitrogenase activity was negligible but protein turnover continued. In their presence nitrogenase activity was greatly stimulated; turnover was also stimulated but to a greater extent in the absence of nitrogenase substrates. The oxyhydrogen reaction also stimulated uptake of ammonium ions by intact filaments in argon in the dark. Only at very low hydrogen tensions can net hydrogen formation be obtained in argon/CO2 in the light, casting considerable doubt on the suitability of hydrogenase-containing organisms for biophotolytic hydrogen formation. Addition of exogenous ammonia to the cultures incubated in argon resulted in a pronounced stimulation of H2 uptake; nitrate and its derivatives had no such effect, nor did various amino acid derivatives of ammonia. 相似文献
10.
Two pathways of hydrogen uptake in Nostoc muscorum are apparent using either oxygen or nitrogen as electron acceptor. Hydrogen uptake (under argon with some oxygen as electron acceptor assayed in the dark; oxyhydrogen reaction) is found to be more active in dense, light-limited cultures than in thin cultures when light is not limiting. Addition of bicarbonate inhibits this hydrogen uptake, because photosynthesis is stimulated. In a cell-free hydrogenase assay, a 10-fold increase of the activity can be measured, after the cells having been kept under lightlimiting conditions. After incubation under light-saturating conditions, no hydrogen uptake is found, when filaments are assayed under argon plus some oxygen. Assaying these cells under a nitrogen atmosphere, a strong hydrogen uptake occurs. The corresponding cell-free hydrogenase assay exhibits low hydrogenase activity. Furthermore, the hydrogen uptake by intact filaments under nitrogen in the light apparently is correlated with nitrogenase activity. These studies give evidence that, under certain physiological conditions, hydrogen uptake of heterocysts proceeds directly via nitrogenase, with no hydrogenase involved.Abbreviations Chl
chlorophyll
- DCMU (diuron)
3-3,4-dichlorophenyl)-1,1-dimethylurea
- pev
packed cell volume 相似文献
11.
The pathways through which NADPH, NADH and H2 provide electrons to nitrogenase were examined in anaerobically isolated heterocysts. Electron donation in freeze-thawed heterocysts and in heterocyst fractions was studied by measuring O2 uptake, acetylene reduction and reduction of horse heart cytochrome c. In freeze-thawed heterocysts and membrane fractions, NADH and H2 supported cyanide-sensitive, respiratory O2 uptake and light-enhanced, cyanide-insensitive uptake of O2 resulting from electron donation to O2 at the reducing side of Photosystem I. Membrane fractions also catalyzed NADH-dependent reduction of cytochrome c. In freeze-thawed heterocysts and soluble fractions from heterocysts, NADPH donated electrons in dark reactions to O2 or cytochrome c through a pathway involving ferredoxin:NADP reductase; these reactions were only slightly influenced by cyanide or illumination. In freeze-thawed heterocysts provided with an ATP-generating system, NADH or H2 supported slow acetylene reduction in the dark through uncoupler-sensitive reverse electron flow. Upon illumination, enhanced rates of acetylene reduction requiring the participation of Photosystem I were observed with NADH and H2 as electron donors. Rapid NADPH-dependent acetylene reduction occurred in the dark and this activity was not influenced by illumination or uncoupler. A scheme summarizing electron-transfer pathways between soluble and membrane components is presented. 相似文献
12.
A comparative study of the development of uptake hydrogenase and nitrogenase activities in cells of the cyanobacterium Anabaena variabilis was performed.
The induction of heterocysts is followed by the induction of both in vivo hydrogen uptake and nitrogenase activities. Interestingly,
a low but significant H2-uptake [2–7 μmoles of H2 · mg−1 (Chl a) · h−1] occurs in cultures with no heterocysts and with no nitrogenase activity. A slight stimulatory effect (30–40%) of H2 on in vivo H2-uptake was observed during the early stages of nitrogenase induction.
However, exogenous H2 does not further stimulate the induction of in vivo hydrogen uptake observed during heterocyst differentiation. Similarly,
organic carbon (fructose) did not influence the induction of either in vivo hydrogen uptake or nitrogenase activities. Exogenous
fructose supports higher in vivo hydrogen uptake and nitrogenase activities when the cells enter late exponential phase of
growth.
Received: 22 November 1995 / Accepted: 22 December 1995 相似文献
13.
Katerina Kourpa Eftychia Manarolaki Alexandros Lyratzakis Vasso Strataki Fiona Rupprecht Julian D. Langer Georgios Tsiotis 《Proteomics》2019,19(19)
Cyanobacteria are oxygenic photosynthetic prokaryotes and play a crucial role in the Earth's carbon and nitrogen cycles. The photoautotrophic cyanobacterium Anabaena sp. PCC 7120 has the ability to fix atmospheric nitrogen in heterocysts and produce hydrogen as a byproduct through a nitrogenase. In order to improve hydrogen production, mutants from Anabaena sp. PCC 7120 are constructed by inactivation of the uptake hydrogenase (ΔhupL) and the bidirectional hydrogenase (ΔhoxH) in previous studies. Here the proteomic differences of enriched heterocysts between these mutants cultured in N2‐fixing conditions are investigated. Using a label‐free quantitative proteomics approach, a total of 2728 proteins are identified and it is found that 79 proteins are differentially expressed in the ΔhupL and 117 proteins in the ΔhoxH variant. The results provide for the first time comprehensive information on proteome regulation of the uptake hydrogenase and the bidirectional hydrogenase, as well as systematic data on the hydrogen related metabolism in Anabaena sp. PCC 7120. 相似文献
14.
The purple sulfur phototrophic bacterium Thiocapsa roseopersicina BBS synthesizes at least three NiFe hydrogenases (Hox, Hup, Hyn). We characterized the physiological H2 consumption/evolution reactions in mutants having deletions of the structural genes of two hydrogenases in various combinations. This made possible the separation of the functionally distinct roles of the three hydrogenases. Data showed that Hox hydrogenase (unlike the Hup and Hyn hydrogenases) catalyzed the dark fermentative H2 evolution and the light-dependent H2 production in the presence of thiosulfate. Both Hox+ and Hup+ mutants demonstrated light-dependent H2 uptake stimulated by CO2 but only the Hup+ mutant was able to mediate O2-dependent H2 consumption in the dark. The ability of the Hox+ mutant to evolve or consume hydrogen was found to depend on a number of interplaying factors including both growth and reaction conditions (availability of glucose, sulfur compounds, CO2, H2, light). The study of the redox properties of Hox hydrogenase supported the reversibility of its action. Based on the results a scheme is suggested to describe the role of Hox hydrogenase in light-dependent and dark hydrogen metabolism in T. roseopersicina BBS. 相似文献
15.
Kinetics of the Oxyhydrogen Reaction in the Presence and Absence of Carbon Dioxide in Scenedesmus obliquus 总被引:2,自引:2,他引:0
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The oxyhydrogen reaction in the presence and absence of CO2 was studied in H2-adapted Scenedesmus obliquus by monitoring the initial rates of H2, O2, and 14CO2 uptake and the effect of inhibitors on these rates with gas-sensing electrodes and isotopic techniques. In the presence of 0.02 atmosphere O2, the pH2 was varied from 0 to 1 atmosphere. Whereas the rate of O2 uptake increased by only 30%, the rate of H2 uptake increased severalfold over the range of pH2 values. At 0.1 atmosphere H2 and 0.02 atmosphere O2, rates for H2 and O2 uptake were between 15 and 25 micromoles per milligram chlorophyll per hour. As the pH2 was changed from 0 to 1 atmosphere, the quotient H2:O2 changed from 0 to roughly 2. This change may reflect the competition between H2 and the endogenous respiratory electron donors. Respiration in the presence of glucose and acetate was also competitive with H2 uptake. KCN inhibited equally respiration (O2 uptake in the absence of H2) and the oxyhydrogen reaction in the presence and absence of CO2. The uncoupler carbonyl cyanide p-trifluoromethoxyphenylhydrazone accelerated the rate of respiration and the oxyhydrogen reaction to a similar extent. It was concluded that the oxyhydrogen reaction both in the presence and absence of CO2 has properties in common with components of respiration and photosynthesis. Participation of these two processes in the oxyhydrogen reaction would require a closely linked shuttle between mitochondrion and chloroplast. 相似文献
16.
Katrin G?rtner Sigal Lechno-Yossef Adam J. Cornish C. Peter Wolk Eric L. Hegg 《Applied and environmental microbiology》2012,78(24):8579-8586
H2 generated from renewable resources holds promise as an environmentally innocuous fuel that releases only energy and water when consumed. In biotechnology, photoautotrophic oxygenic diazotrophs could produce H2 from water and sunlight using the cells'' endogenous nitrogenases. However, nitrogenases have low turnover numbers and require large amounts of ATP. [FeFe]-hydrogenases found in other organisms can have 1,000-fold higher turnover numbers and no specific requirement for ATP but are very O2 sensitive. Certain filamentous cyanobacteria protect nitrogenase from O2 by sequestering the enzyme within internally micro-oxic, differentiated cells called heterocysts. We heterologously expressed the [FeFe]-hydrogenase operon from Shewanella oneidensis MR-1 in Anabaena sp. strain PCC 7120 using the heterocyst-specific promoter PhetN. Active [FeFe]-hydrogenase was detected in and could be purified from aerobically grown Anabaena sp. strain PCC 7120, but only when the organism was grown under nitrate-depleted conditions that elicited heterocyst formation. These results suggest that the heterocysts protected the [FeFe]-hydrogenase against inactivation by O2. 相似文献
17.
Following NTG mutagenesis, four independent mutants of Anabaena PCC7120 defective in heterocyst differentiation were isolated. These fell into 2 distinct classes; (1) those unable to differentiate heterocysts or show whole-cell acetylene reduction activity; and (2) those unable to differentiate heterocysts but capable of microaerobic acetylene reduction. All mutants grew equally well as the wild type with added nitrogen sources and showed no apparent differences in glutamine synthetase or glutamate synthase activities compared with the wild type. The mutants of class (2) evolved H2 only under microaerobic conditions, suggesting that H2 is evolved via nitrogenase in Anabaena PCC7120. 相似文献
18.
Simultaneous measurement of oxygen and hydrogen exchange from the blue-green alga anabaena 总被引:16,自引:4,他引:12
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Two Clark-type polarographic electrodes were used to measure simultaneous H2 and O2 exchange from three species of the blue-green alga Anabaena. Maximum H2 photoevolution from N2-fixing cultures of Anabaena required only the removal of dissolved O2 and N2; no adaptation period was necessary. No correlation of H2 photoproduction with photosynthetic O2 evolution, beyond their mutual light requirement, was found. Hydrogen photoevolution has the following characteristics in common with N2 fixation in these organisms: DCMU insensitivity; similar white light dependency with very low dark production rates; maximum efficiency in photosystem I light; inhibition by N2, O2 and acetylene; and an apparent requirement for the presence of heterocysts. Growth on nitrate medium reduces, and on ammonium medium obliterates, both reactions. Cultures grown under limiting CO2 conditions have H2 photoproduction rates proportional to their growth rates. Hydrogenase activity is inferred from H2 uptake in the dark, but this activity apparently is independent of the photoevolution of H2 which is ascribed strictly to the nitrogenase system. 相似文献
19.
Physiological Studies of Oxygen Protection Mechanisms in the Heterocysts of Anabaena cylindrica 总被引:9,自引:0,他引:9
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The mechanism of O2 protection of nitrogenase in the heterocysts of Anabaena cylindrica was studied in vivo. Resistance to O2 inhibition of nitrogenase activity correlated with the O2 tension of the medium in which heterocyst formation was induced. O2 resistance also correlated with the apparent Km for acetylene, indicating that O2 tension may influence the development of a gas diffusion barrier in the heterocysts. The role of respiratory activity in protecting nitrogenase from O2 that diffuses into the heterocyst was studied using inhibitors of carbon metabolism. Reductant limitation induced by 3-(3,4-dichlorophenyl)-1, 1-dimethylurea increased the O2 sensitivity of in vivo acetylene reduction. Azide, at concentrations (30 mM) sufficient to completely inhibit dark nitrogenase activity (a process dependent on oxidative phosphorylation for its ATP supply), severely inhibited short-term light-dependent acetylene reduction in the presence of O2 but not in its absence. After 3 h of aerobic incubation in the presence of 20 mM azide, 75% of cross-reactive component I (Fe-Mo protein) in nitrogenase was lost; less than 35% was lost under microaerophilic conditions. Sodium malonate and monofluoroacetate, inhibitors of Krebs cycle activity, had only small inhibitory effects on nitrogenase activity in the light and on cross-reactive material. The results suggest that oxygen protection is dependent on both an O2 diffusion barrier and active respiration by the heterocyst. 相似文献
20.
Sergey Kosourov Hannu Leino Gayathri Murukesan Fiona Lynch Kaarina Sivonen Anatoly A. Tsygankov Eva-Mari Aro Yagut Allahverdiyeva 《Applied and environmental microbiology》2014,80(18):5807-5817
We have investigated two approaches to enhance and extend H2 photoproduction yields in heterocystous, N2-fixing cyanobacteria entrapped in thin alginate films. In the first approach, periodic CO2 supplementation was provided to alginate-entrapped, N-deprived cells. N deprivation led to the inhibition of photosynthetic activity in vegetative cells and the attenuation of H2 production over time. Our results demonstrated that alginate-entrapped ΔhupL cells were considerably more sensitive to high light intensity, N deficiency, and imbalances in C/N ratios than wild-type cells. In the second approach, Anabaena strain PCC 7120, its ΔhupL mutant, and Calothrix strain 336/3 films were supplemented with N2 by periodic treatments of air, or air plus CO2. These treatments restored the photosynthetic activity of the cells and led to a high level of H2 production in Calothrix 336/3 and ΔhupL cells (except for the treatment air plus CO2) but not in the Anabaena PCC 7120 strain (for which H2 yields did not change after air treatments). The highest H2 yield was obtained by the air treatment of ΔhupL cells. Notably, the supplementation of CO2 under an air atmosphere led to prominent symptoms of N deficiency in the ΔhupL strain but not in the wild-type strain. We propose that uptake hydrogenase activity in heterocystous cyanobacteria not only supports nitrogenase activity by removing excess O2 from heterocysts but also indirectly protects the photosynthetic apparatus of vegetative cells from photoinhibition, especially under stressful conditions that cause an imbalance in the C/N ratio in cells. 相似文献