A new,fast, and sensitive assay for NADH-ferredoxin oxidoreductase detection in clostridia

A new, simple and very sensitive assay for NADH-ferredoxin or flavodoxin reductase activity is described. The assay is based on the nonenzymatic reduction of the metronidazole by ferredoxin or flavodoxin. In the presence of NADH, ferredoxin or flavodoxin and cell-free extract of clostridia, no metronidazole reduction is observed; the reaction occurs only if acetyl-CoA is added to the reaction mixture. Metronidazole reduction is quantitated by the spectrophotometric measurement at 320 nm. In this assay the change in absorbance is linearly related to the amount of clostridial extract for concentration of 0.1 to 0.8 mg/ml and to the flavodoxin or ferredoxin for concentrations of 0.5 to 8 nmol/ml.     

The path of electron transfer to nitrogenase in a phototrophic alpha‐proteobacterium

The phototrophic alpha‐proteobacterium, Rhodopseudomonas palustris, is a model for studies of regulatory and physiological parameters that control the activity of nitrogenase. This enzyme produces the energy‐rich compound H₂, in addition to converting N₂ gas to NH₃. Nitrogenase is an ATP‐requiring enzyme that uses large amounts of reducing power, but the electron transfer pathway to nitrogenase in R. palustris was incompletely known. Here, we show that the ferredoxin, Fer1, is the primary but not sole electron carrier protein encoded by R. palustris that serves as an electron donor to nitrogenase. A flavodoxin, FldA, is also an important electron donor, especially under iron limitation. We present a model where the electron bifurcating complex, FixABCX, can reduce both ferredoxin and flavodoxin to transfer electrons to nitrogenase, and we present bioinformatic evidence that FixABCX and Fer1 form a conserved electron transfer pathway to nitrogenase in nitrogen‐fixing proteobacteria. These results may be useful in the design of strategies to reroute electrons generated during metabolism of organic compounds to nitrogenase to achieve maximal activity.     

Consequences of the iron-dependent formation of ferredoxin and flavodoxin on photosynthesis and nitrogen fixation on Anabaena strains

Iron-dependent formation of ferredoxin and flavodoxin was determined in Anabaena ATCC 29413 and ATCC 29211 by a FPLC procedure. In the first species ferredoxin is replaced by flavodoxin at low iron levels in the vegetative cells only. In the heterocysts from Anabaena ATCC 29151, however, flavodoxin is constitutively formed regardless of the iron supply.Replacement of ferredoxin by flavodoxin had no effect on photosynthetic electron transport, whereas nitrogen fixation was decreased under low iron conditions. As ferredoxin and flavodoxin exhibited the same K_m values as electron donors to nitrogenase, an iron-limited synthesis of active nitrogenase was assumed as the reason for inhibited nitrogen fixation. Anabaena ATCC 29211 generally lacks the potential to synthesize flavodoxin. Under iron-starvation conditions, ferredoxin synthesis is limited, with a negative effect on photosynthetic oxygen evolution.     

Utilization of free and immobilized Desulfovibrio hydrogenase in hydrogen photoproduction : Coupling efficiency of cytochrome C3, ferredoxin and flavodoxin

Summary Hydrogen photoproduction has been achieved by coupling free or immobilized hydrogenases from Desulfovibrio species to illuminated chloroplasts through different electron mediators. Whereas D. gigas flavodoxin or ferredoxin I cannot directly mediate the electron flux from chloroplasts to hydrogenase, the addition of these mediators considerably enhances the H₂ photoproduction of a system including cytochrome C₃. These immobilized hydrogenases exhibit good stability under working conditions and can be re-used.     

Hydrogen peroxide synthesis in isolated spinach chloroplast lamellae : an analysis of the mehler reaction in the presence of NADP reduction and ATP formation

Light-dependent O₂ reduction concomitant with O₂ evolution, ATP formation, and NADP reduction were determined in isolated spinach (Spinacia oleracea L. var. America) chloroplast lamellae fortified with NADP and ferredoxin. These reactions were investigated in the presence or absence of catalase, providing a tool to estimate the reduction of O₂ to H₂O₂ (Mehler reaction) concomitant with NADP reduction. In the presence of 250 micromolar O₂, O₂ photoreduction, simultaneous with NADP photoreduction, was dependent upon light intensity, ferredoxin, Mn²⁺, NADP, and the extent of coupling of phosphorylation to electron flow.

In the presence of an uncoupling concentration of NH₄⁺, saturating light intensity (>500 watts/square meter), saturating ferredoxin (10 micromolarity) rate-limiting to saturating NADP (0.2-0.9 millimolarity), and Mn²⁺ (50-1000 micromolarity), the maxium rates of O₂ reduction were 13-25 micromoles/milligram chlorophyll per hour, while concomitant rates of O₂ evolution and NADP reduction were 69 to 96 and 134 to 192 micromoles/milligram chlorophyll per hour, respectively. Catalase did not affect the rate of NADPH or ATP formation but decreased the NADPH:O₂ ratios from 2.3-2.8 to 1.9-2.1 in the presence of rate-limiting as well as saturating concentrations of NADP.

Photosynthetic electron flow at a rate of 31 micromoles O₂ evolved/milligram chlorophyll per hour was coupled to the synthesis of 91 micromoles ATP/milligram chlorophyll per hour, while the concomitant rate of O₂ reduction was 0.6 micromoles/milligram chlorophyll per hour and was calculated to be associated with an apparent ATP formation of only 2 micromoles/milligram chlorophyll per hour. Thus, electron flow from H₂O to O₂ did not result in ATP formation significantly above that produced during NADP reduction.

     

Molecular weight and quaternary structure of ribulose bisphosphate carboxylase from the cyanobacterium,Synechococcus sp.

Inhibition of photosynthetic growth of Rhodopseudomonas capsulata by metronidazole was dependent on the nitrogen supply in culture solutions. Cultures fixing dinitrogen were more susceptible to inhibition by low concentrations than those supplied with NH ₄ ⁺ . Light-dependent C₂H₂ reduction and H₂ production by washed cells were inhibited by 80% and 60% respectively by 1 mM metronidazole. When this compound was first reduced with H₂-palladised asbestos prior to assay, it only partially restricted C₂H₂ reduction in washed cells (33%) compared with unreduced inhibitor (68%). Metronidazole was without effect on other metabolic functions. Thus, even at 40 mM it did not inhibit either (a) dark or light respiration in cells grown under photo- and chemo-heterotrophic conditions; (b) H₂-dependent photoreduction of ¹⁴CO₂; (c) -glutamyltransferase activity of glutamine synthetase in cell-free extracts (25 mM inhibitor).Metronidazole (1 mM) completely inhibited C₂H₂ reduction by washed cells of Azotobacter vinelandii. The dithionite-dependent C₂H₂ reduction of a partially purified nitrogenase was only partially inhibited (30%) by 1 mM metronidazole.     

Metabolism in hyperthermophilic microorganisms

Hyperthermophilic microorganisms grow at temperatures of 90 °C and above and are a recent discovery in the microbial world. They are considered to be the most ancient of all extant life forms, and have been isolated mainly from near shallow and deep sea hydrothermal vents. All but two of the nearly twenty known genera are classified asArchaea (formerly archaebacteria). Virtually all of them are strict anaerobes. The majority are obligate heterotrophs that utilize proteinaceous materials as carbon and energy sources, although a few species are also saccharolytic. Most also depend on the reduction of elemental sulfur to hydrogen sulfide (H₂S) for significant growth. Peptide fermentation involves transaminases and glutamate dehydrogenase, together with several unusual ferredoxin-linked oxidoreductases not found in mesophilic organisms. Similarly, a novel pathway based on a partially non-phosphorylated Entner-Doudoroff scheme has been postulated to convert carbohydrates to acetate, H₂ and CO₂, although a more conventional Embden-Meyerhof pathway has also been identified in one saccharolytic species. The few hyperthermophiles known that can assimilate CO₂ do so via a reductive citric acid cycle. Two S^o-reducing enzymes termed sulfhydrogenase and sulfide dehydrogenase have been purified from the cytoplasm of a hyperthermophile that is able to grow either with or without S^o. A scheme for electron flow during the oxidation of carbohydrates and peptides and the reduction of S^o has been proposed. However, the mechanisms by which S^o reduction is coupled to energy conservation in this organism and in obligate S^o-reducing hyperthermophiles is not known.Abbreviations ADH alcohol dehydrogenase (ADH) - AOR aldehyde ferredoxin oxidoreductase - FMOR formate ferredoxin oxidoreductase - FOR formaldehyde ferredoxin oxidoreductase - GAPDH glyceraldehyde-3-phosphate dehydrogenase - GDH glutamate dehydrogenase - GluOR glucose ferredoxin oxidoreductase - KGOR 2-ketoglutarate ferredoxin oxidoreductase - IOR indolepyruvate ferredoxin oxidoreductase - LDH lactate dehydrogenase - MPT molybopterin - POR pyruvate ferredoxin oxidoreductase - PLP pyridoxal-phosphate - PS polysulfide - TPP thiamin pyrophosphate - S^o elemental sulfur - VOR isovalerate ferredoxin oxidoreductase     

Regulation and function of ferredoxin-linked versus cytochrome b-linked hydrogenase in electron transfer and energy metabolism of Methanosarcina barkeri MS

Acetate-grown cells of Methanosarcina barkeri MS were found to form methane from H₂:CO₂ at the same rate as hydrogen-grown cells. Cells grown on acetate had similar levels of soluble F₄₂₀-reactive hydrogenase I, and higher levels of cytochrome-linked hydrogenase II compared to hydrogen-grown cells. The hydrogenase I and II activities in the crude extract of acetate-grown cells were separated by differential binding properties to an immobilized Cu²⁺ column. Hydrogenase II did not react with ferredoxin or F₄₂₀, whereas hydrogenase I coupled to both ferredoxin and F₄₂₀. A reconstituted soluble protein system composed of purified CO dehydrogenase, F₄₂₀-reactive hydrogenase I fraction, and ferredoxin produced H₂ from CO oxidation at a rate of 2.5 nmol/min · mg protein. Membrane-bound hydrogenase II coupled H₂ consumption to the reduction of CoM-S-S-HTP and the synthesis of ATP. The differential function of hydrogenase I and II is ascribed to ferredoxin-linked hydrogen production from CO and cytochrome b-linked H₂ consumption coupled to methanogenesis and ATP synthesis, respectively.     

The Bidirectional NiFe-hydrogenase in Synechocystis sp. PCC 6803 Is Reduced by Flavodoxin and Ferredoxin and Is Essential under Mixotrophic,Nitrate-limiting Conditions

Cyanobacteria are able to use solar energy for the production of hydrogen. It is generally accepted that cyanobacterial NiFe-hydrogenases are reduced by NAD(P)H. This is in conflict with thermodynamic considerations, as the midpoint potentials of NAD(P)H do not suffice to support the measured hydrogen production under physiological conditions. We show that flavodoxin and ferredoxin directly reduce the bidirectional NiFe-hydrogenase of Synechocystis sp. PCC 6803 in vitro. A merodiploid ferredoxin-NADP reductase mutant produced correspondingly more photohydrogen. We furthermore found that the hydrogenase receives its electrons via pyruvate:flavodoxin/ferredoxin oxidoreductase (PFOR)-flavodoxin/ferredoxin under fermentative conditions, enabling the cells to gain ATP. These results strongly support that the bidirectional NiFe-hydrogenases in cyanobacteria function as electron sinks for low potential electrons from photosystem I and as a redox balancing device under fermentative conditions. However, the selective advantage of this enzyme is not known. No strong phenotype of mutants lacking the hydrogenase has been found. Because bidirectional hydrogenases are widespread in aquatic nutrient-rich environments that are capable of triggering phytoplankton blooms, we mimicked those conditions by growing cells in the presence of increased amounts of dissolved organic carbon and dissolved organic nitrogen. Under these conditions the hydrogenase was found to be essential. As these conditions close the two most important sinks for reduced flavodoxin/ferredoxin (CO₂-fixation and nitrate reduction), this discovery further substantiates the connection between flavodoxin/ferredoxin and the NiFe-hydrogenase.     

Differential activities of heterocyst ferredoxin,vegetative cell ferredoxin,and flavodoxin as electron carriers in nitrogen fixation and photosynthesis in Anabaena sp.

In cyanobacteria an increasing number of low potential electron carriers is found, but in most cases their contribution to metabolic pathways remains unclear. In this work, we compare recombinant plant-type ferredoxins from Anabaena sp. PCC 7120, encoded by the genes petF and fdxH, respectively, and flavodoxin from Anabaena sp. PCC 7119 as electron carriers in reconstituted in vitro assays with nitrogenase, Photosystem I, ferredoxin-NADP⁺ reductase and pyruvate-ferredoxin oxidoreductase. In every experimental system only the heterocyst ferredoxin catalyzed an efficient electron transfer to nitrogenase while vegetative cell ferredoxin and flavodoxin were much less active. This implies that flavodoxin is not able to functionally replace heterocyst ferredoxin. When PFO-activity in heterocyst extracts was reconstituted under anaerobic conditions, both ferredoxins were more efficient than flavodoxin, which suggested that this PFO was of the ferredoxin dependent type. Flavodoxin, synthesized under iron limiting conditions, replaces PetF very efficiently in the electron transport from Photosystem I to NADP⁺, using thylakoids from vegetative cells.Abbreviations BSA bovine serum albumin - FdxH heterocyst ferredoxin - Fld flavodoxin - FNR ferredoxin-NADP⁺ reductase - MV methyl viologen - PetF vegetative cell ferredoxin - PFO pyruvate-ferredoxin oxidoreductase - Pyr piruvate - PS I Photosystem I     

Immunoquantification of flavodoxin and ferredoxin from Scenedesmus vacuolatus (Chlorophyta) as iron-stress molecular markers

Quantification of the iron nutritional status of phytoplankton is of great interest not only for the study of the oceans but also for fresh waters. Flavodoxin is a small flavoprotein proposed as a molecular marker for iron stress, since it is induced as a consequence of iron deprivation, replacing the iron-sulphur protein ferredoxin. Flavodoxin and ferredoxin from Scenedesmus vacuolatus have been immunoquantified in cells grown under different iron nutritional conditions. Flavodoxin and ferredoxin levels correlate with the iron availability, and the calculated flavodoxin index can be used as an iron-stress marker. Other physiological parameters such as copper deficiency, heterotrophic or mixotrophic growth, nitrogen source and salt stress were also tested as potential factors influencing flavodoxin expression. Salt stress and heterotrophic growth conditions alter flavodoxin and ferredoxin expression. Once flavodoxin expression is repressed by iron (and severe deficiency alleviated), S.vacuolatus still increases its ferredoxin from 0·5 to 1·6 mol of ferredoxin per mole of ferredoxin-NADP⁺ reductase, and this ratio can be used for the evaluation of mild deficiency.     

Construction and characterization of an Azotobacter vinelandii strain with mutations in the genes encoding flavodoxin and ferredoxin I.

Flavodoxin and ferredoxin I have both been implicated as components of the electron transport chain to nitrogenase in the aerobic bacterium Azotobacter vinelandii. Recently, the genes encoding flavodoxin (nifF) and ferredoxin I (fdxA) were cloned and sequenced and mutants were constructed which are unable to synthesize either flavodoxin (DJ130) or ferredoxin I (LM100). Both single mutants grow at wild-type rates under N2-fixing conditions. Here we report the construction of a double mutant (DJ138) which does not synthesize either flavodoxin or ferredoxin I. When plated on ammonium-containing medium, this mutant had a very small colony size when compared with the wild type, and in liquid culture with ammonium, this double mutant grew three times slower than the wild type or single mutant strains. This demonstrated that there is an important metabolic function unrelated to nitrogen fixation that is normally carried out by either flavodoxin or ferredoxin. If either one of these proteins is missing, the other can substitute for it. The double mutant phenotype can now be used to screen site-directed mutant versions of ferredoxin I for functionality in vivo even though the specific function of ferredoxin I is still unknown. The double mutant grew at the same slow rate under N2-fixing conditions. Thus, A. vinelandii continues to fix N2 even when both flavodoxin and ferredoxin I are missing, which suggests that a third as yet unidentified protein also serves as an electron donor to nitrogenase.     

Laser-flash absorption spectroscopy study of the competition between ferredoxin and flavodoxin photoreduction by Photosystem I in Synechococcus sp. PCC 7002: Evidence for a strong preference for ferredoxin

The competition between ferredoxin and flavodoxin for electrons from Photosystem I was analyzed by flash absorption spectroscopy of the photoreduction processes that take place in the presence of both acceptor proteins in vitro. Steady state photoreduction assays indicate a strong inhibition of the apparent flavodoxin photoreduction activities of Photosystem I in the presence of ferredoxin. Flash-absorption experiments carried out at 626 nm, a wavelength where the reduction of ferredoxin shows no spectral contribution, show that the photoreduction of oxidized flavodoxin and flavodoxin semiquinone are inhibited by ferredoxin in a quantitatively similar way. The experimental data can be satisfactorily described by a reaction model that assumes that both redox states of flavodoxin do not compete with ferredoxin for binding on PS I and that the binding equilibrium between ferredoxin and PS I is not changed in their presence. In contrast, a model which assumes that ferredoxin and flavodoxin actually compete for binding to PS I gives poor results. Similarly, experimental data observed in the presence of both redox states of flavodoxin can also be quantitatively described under the assumption that the binding equilibrium between flavodoxin semiquinone and PS I is not disturbed by oxidized flavodoxin. Taken together, this analysis shows that PS I favors ferredoxin over flavodoxin and flavodoxin semiquinone over oxidized flavodoxin. This behavior is in accordance with the values of the dissociation constants for complexes between PS I and its acceptors. However, in case of ferredoxin the observed preference is stronger than expected from these values, indicating that ferredoxin is almost absolutely preferred by PS I over flavodoxin and is always reduced first.     

Estradiol attenuation of β-amyloid-induced toxicity: A comparison of MTT and calcein AM assays

17β-estradiol (βE2) has been shown to attenuate the toxicity of β-amyloid peptides (Aβ) in neuronal cultures with the effective concentration of βE2 ranging from low nM to high μM. This study compares the effective neuroprotective concentration of βE2 against both Aβ-mediated toxicity in a human neuroblastoma cell line, SK-N-SH using cellular reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) as an endpoint to the effective βE2 concentration obtained using a calcein acetoxymethyl ester (calcein AM) viability assay. The minimum βE2 concentration required for protection varied 1000-fold between the two viability assays with 1 nM βE2 conferring significant protection in the calcein AM assay but 1 μM βE2 required for significant protection in the MTT assay. Interestingly, the maximal inhibition of MTT reduction occured at sub-toxic Aβ concentrations and did not correlate with other markers of cellular viability including calcein fluorescence, dye exclusion (propidium iodide or trypan blue), cellular ATP levels, or reduction of another tetrazolium dye, 5-(3-carboxymethoxyphenyl)-2-(4,5-dimethylthiazolyl)-3-(4-sulfophenyl) tetrazolium (MTS). By contrast, there was no difference between the MTT and calcein AM assays with respect to H₂O₂ toxicity or the neuroprotective effectiveness of 10 nM βE2 against H_{2 2} toxicity. These results indicate that low concentrations of βE2 can attenuate Aβ and H₂O₂ toxicity in a human neuroblastoma cell line. Further, these results suggest that the MTT assay is not an appropriate assay for the determination of βE2-mediated attenuation of Aβ toxicity.     

Properties and regulation of synthesis of two ferredoxins from Rhodopseudomonas capsulata

Two ferredoxins from nitrogen-fixing cells of the phototrophic bacterium Rhodopseudomonas capsulata, strain B10, are purified to a homogeneous state and characterized. The molecular mass of ferredoxin I is about 12 kDa and that of ferredoxin II, 18 kDa. Ferredoxin I contains 8 Fe²⁺ and 8 S^2?; ferredoxin II has 4 Fe²⁺ and 4 S^2? per molecule. The redox potential of ferredoxin I is about ?270 mV and that of ferredoxin II ?419 mV. Ferredoxin I is more labile to the action of O₂, O^?₂, H₂O₂ and heating. The ferredoxins are also different in their absorption and EPR spectra, amino acid composition and electron-transfer activity to Rps. capsulata nitrogenase: both C₂H₂ reduction and H₂ evolution by Rps. capsulata nitrogenase proceed faster in the presence of ferredoxin I than in case of ferredoxin II. Synthesis of ferredoxin I takes place only in Rps. capsulata nitrogen-fixing cells grown in light under anaerobic conditions whereas ferredoxin II formation does not depend on the source of nitrogen or the growth medium, though the amount of ferredoxin II varies with the growth conditions. Its highest level has been found in the cells grown in lactate-limited medium in the presence of CO₂ and light or in the presence of glutamate in darkness under anaerobic conditions.     

Chloroplast Respiration : A MEANS OF SUPPLYING OXIDIZED PYRIDINE NUCLEOTIDE FOR DARK CHLOROPLASTIC METABOLISM

A spinach (Spinacia oleracia var. America) chloroplast particle fortified with ferredoxin, fructose-1,6-bisphosphate, or ribose-5-phosphate and NADP has been shown to generate NADPH by the oxidation of glyceraldehyde-3 phosphate to glycerate-3-phosphate (PGA) and to reduce ferredoxin with the NADPH. The resulting reduced ferredoxin can reduce O₂ to H₂O₂, nitrite to ammonia, or protons to H₂. Hydrogen production was the result of adding hydrogenase from Chlamydomonas reinhardii to the chloroplast preparation. The predicted stoichiometry of 1 PGA:1 O₂ in the absence of and 2 PGA:1 O₂ in the presence of catalase was observed indicating H₂O₂ as the end product of O₂ reduction. The predicted stoichiometry of 3 PGA:1 nitrite:1 ammonia was also observed. A scheme is presented to account for a sustained generation of NADP and ATP necessary for the dissimilation of starch in the darkened chloroplast. The unifying term chloroplast respiration is introduced to account for those reactions in which reduced ferredoxin interacts with physiological acceptors other than NADP or nitrite, hydrogen, or O₂ respiration when nitrite, protons, or O₂ is the ultimate electron acceptor.     

Relationship between P700 turnover (m second) and NADP reduction as a function of ferredoxin concentration in isolated broken chloroplasts

Steady-state electron flux through P₇₀₀ (t $\text{1}\text{2}$ 20 msec) and concomitant rate of NADP reduction have been measured under weak actinic illumination as a function of concentration of ferredoxin added to broken chloroplasts isolated from peas. At suboptimal concentrations of ferredoxin this P₇₀₀ is not sufficient to account for the NADP reduction. At high concentrations ferredoxin inhibits the rate of NADP reduction without affecting the P₇₀₀ flux under short wavelength illumination. Under far red illumination P₇₀₀ flux is also inhibited by ferredoxin at high concentrations. Addition of 5 mM Mg⁺⁺ increases the rate of NADP reduction at all concentrations of ferredoxin under both kinds of illumination, while P₇₀₀ flux is inhibited under short wavelength illumination and remains unchanged under far red illumination. The results indicate that the observed (20 msec) P₇₀₀ is not involved in NADP reduction.     

Hydrogenase Activity and the H2-Fumarate Electron Transport System in Bacteroides fragilis

Hydrogenase activity and the H₂-fumarate electron transport system in a carbohydrate-fermenting obligate anaerobe, Bacteroides fragilis, were investigated. In both whole cells and cell extracts, hydrogenase activity was demonstrated with methylene blue, benzyl viologen, flavin mononucleotide, or flavin adenine dinucleotide as the electron acceptor. A catalytic quantity of benzyl viologen or ferredoxin from Clostridium pasteurianum was required to reduce nicotinamide adenine dinucleotide or nicotinamide adenine dinucleotide phosphate with H₂. Much of the hydrogenase activity appeared to be associated with the soluble fraction of the cell. Fumarate reduction to succinate by H₂ was demonstrable in cell extracts only in the presence of a catalytic quantity of benzyl viologen, flavin mononucleotide, flavin adenine dinucleotide, or ferredoxin from C. pasteurianum. Sulfhydryl compounds were not required for fumarate reduction by H₂, but mercaptoethanol and dithiothreitol appeared to stimulate this activity by 59 and 61%, respectively. Inhibition of fumarate reduction by acriflavin, rotenone, 2-heptyl-4-hydroxyquinoline-N-oxide, and antimycin A suggest the involvement of a flavoprotein, a quinone, and cytochrome b in the reduction of fumarate to succinate. The involvement of a quinone in fumarate reduction is also apparent from the inhibition of fumarate reduction by H₂ when cell extracts were irradiated with ultraviolet light. Based on the evidence obtained, a possible scheme for the flow of electrons from H₂ to fumarate in B. fragilis is proposed.     

The pyruvate: Ferredoxin oxidoreductase in heterocysts of the cyanobacteriuim Anabaena cylindrica

Heterocyst preparations have been obtained which actively perform nitrogen fixation (C₂H₂ reduction) and contain the enzymes of glycolysis and some of the tricarboxylic acid cycle. Pyruvate: ferredoxin oxidereductase has been unambiguously demonstrated in extracts from heterocysts by the formation of acetylcoenzyme A, CO₂ and reduced methyl viologen (ferredoxi) from pyruvate, coenzyme A and oxidized methyl viologen (ferredoxin) as well as by the synthesis of pyruvate from CO₂, acetylcoenzyme A and reduced methyl viologen. Pyruvate supports C₂H₂ reduction by isolated heterocysts, however, with lower activity than Na₂S₂O₄ and H₂. α-Ketoglutarate: ferredoxin oxidoreductase is absent in Anabaena cylindrica, confirming that the organism has an incomplete tricarboxylic acid cycle.