Nitrite and hydroxylamine reduction in higher plants. Fractionation, electron donor and substrate specificity of leaf enzymes, principally from vegetable marrow (Cucurbita pepo L.)

Nitrite reductase was purified between 760- and 1300-fold from vegetable marrow (Cucurbita pepo L.) and residual hydroxylamine reductase activity was low or negligible by comparison. With ferredoxin as electron donor, nitrite loss and ammonia formation at pH7.5 were stoicheiometrically equivalent. Crude nitrite reductase preparations showed negligible activity with NADPH as electron donor maintained in the reduced state by glucose 6-phosphate, whereas by comparison, activity was high when either ferredoxin or benzyl viologen were also present and reduced by the NADPH-glucose 6-phosphate system, whereas FMNH(2) produced variable and relatively low activity under the same conditions. At pH values below 7, non-enzymic reactions occurred between reduced benzyl viologen and nitrite, and intermediate reduction products were inferred to be produced instead of ammonia. Activity with ferredoxin (0.1mm), reduced by chloroplast grana in the light, was 25 times that produced with ferredoxin (40mum) reduced with NADPH and glucose 6-phosphate. For an approximate molecular weight 61000-63000 derived by chromatography on Sephadex G-100 and G-200, and a specific activity of 46mumol of nitrite reduced/min per mg of protein with light and chloroplast grana, a minimum turnover number of 3x10(3)mol of nitrite reduced/min per mol of enzyme was found. Two hydroxylamine reductases were separated on Sephadex gels. One (HR1) was initially associated with nitrite reductase during gel filtration but disappeared during later fractionation. This HR1 fraction showed nearly comparable activity with reduced benzyl viologen, ferredoxin or FMNH(2). The other (HR2), of molecular weight approx. 35000, reacted with reduced benzyl viologen but showed negligible activity with ferredoxin or NADPH. Activity with FMNH(2) was associated with an irregular trailing boundary during gel filtration, with much diminished activity in the HR2 region. Activity with NADPH was about 30% of that with FMNH(2), reduced benzyl viologen or ferredoxin and was considered to reside in fraction HR1. Hydroxylamine yielded ammonia under all assay conditions. No activity with hyponitrite or sulphite was observed with reduced benzyl viologen as electron donor in either the nitrite reductase or the hydroxylamine reductase systems, but pyruvic oxime produced about 4% of the activity of hydroxylamine.     

Purification and Properties of Nitrite Reductase from Spinach Leaves

Nitrite reductase (EC 1.6.6.4) has been purified 730-fold from spinach leaves. The enzyme catalyzes the reduction of nitrite to ammonia, with the use of reduced form of methyl viologen and ferredoxin. A stoichiometry of one molecule of nitrite reduced per molecule of ammonia formed has been found. KCN at 2.5×10^-4 m inhibited nitrite reductase activity almost completely. Purified enzyme was almost homogeneous by disk electrophoresis with polyacrylamide gel. The molecular weight of the enzyme was estimated to be 61,000 from gel filtration. Nitrite reductase, in the oxidized form, has absorption maxima at 276, 388 and 573 mμ. Both methyl viologen and ferredoxin linked nitrite reductase activities of the enzyme were inactivated on exposure to low ionic strength.     

Further Characterization of Ferredoxin Nitrite Reductase and the Relationship between the Enzyme and Methyl Viologen-dependent Nitrite Reductase

Ferredoxin-dependent nitrite reductase of spinach has been further characterized and the relationship between this enzyme and methyl viologen-dependent nitrite reductase studied.

Purified ferredoxin nitrite reductase, having a molecular weight of 86,000, showed 2.5 times higher ferredoxin-dependent activity than methyl viologen-linked activity. Besides 4 mol of labile sulfide the enzyme contained about 2 mol of siroheme per mol. When dithionite, methyl viologen and nitrite were added, ESR signals of a heme nitrosyl complex at g = 2.14, 2.07 and 2.02 were observed. Moreover, hyperfine splitting of the signal due to ¹⁴N nuclear spin was also observed at 2.033, 2.023 and 2.013. The sole addition of hydroxylamine to the ferric enzyme also caused the same but much less intense signals with the hyperfine splitting.

On treatment of the ferredoxin nitrite reductase (native enzyme) with DEAE-Sephadex A-50 chromatography, a modified nitrite reductase having a molecular weight of 61,000 and a protein fraction having an apparent molecular weight of 24,000 were separated. The modified enzyme contained about one mol of siroheme and 4 mol of labile sulfide per mol and showed essentially the same heme ESR signals as the native enzyme. Contrary to the native enzyme, this modified enzyme accepted electrons more efficiently from methyl viologen than ferredoxin and the reduction of nitrite to ammonia catalyzed by the modified enzyme was not stoichiometric. The observed nitrite to ammonia ratio was 1 to less than 0.6. Cyanide at concentrations between 0.02 to 0.2 mm inhibited the activity of the native enzyme almost completely but the modified enzyme was inhibited only partially.

From the results obtained, it is suggested that the native ferredoxin-linked nitrite reductase consists of two components (or subunits) and removal of the light component results in formation of a modified enzyme with increased relative affinity to methyl viologen.     

The purification and properties of nitrite reductase from higher plants, and its dependence on ferredoxin

1. NADPH-dependent nitrite reductase from the leaves of higher plants was purified at least 70-fold and separated into two enzyme fractions. The first enzyme, a diaphorase with ferredoxin-NADP-reductase activity, is required only to transfer electrons from NADPH to a suitable electron acceptor, which then donates electrons to nitrite reductase proper. 2. Purified nitrite reductase accepted electrons from ferredoxin (the natural donor) or from reduced dyes. Ferredoxin was reduced by illuminated chloroplasts or dithionite, or by NADPH when diaphorase was present. The purified enzyme did not accept electrons directly from NADPH. 3. Ferredoxins purified from maize, spinach or Clostridium were interchangeable in the nitrite-reductase system. 4. Nitrite reductase had K(m) 0.15mm for nitrite. The pH optimum varied with plant and method of assay. The preparation had low sulphite-reductase activity. Ammonia was the product of nitrite reduction. 5. For some plants, the assay of crude preparations with NADPH was limited by diaphorase and the addition of diaphorase gave a better estimate of nitrite-reductase activity. A simple method of assay is described that uses dithionite with benzyl viologen as electron donor.     

Events Surrounding the Early Development of Euglena Chloroplasts: VII. Photocontrol of the Source Of Reducing Power for Chloramphenicol Reduction by the Ferredoxin-NADP Reductase System

d(-)threo-Chloramphenicol blocks chlorophyll and plastid protein synthesis in Euglena. During chloroplast development in white light, but not in red, the cells escape from chloramphenicol inhibition and chlorophyll formation is restored. Concomitantly, chloramphenicol is reduced. Reduction of chloramphenicol in an enzyme extract from Euglena requires NADPH and ferredoxin for maximal activity. Methyl viologen replaces ferredoxin, and when chemically reduced, ferredoxin or methyl viologen reduces chloramphenicol directly. This suggests that the enzyme involved is ferredoxin-NADP reductase. In agreement, crude extracts from wild type and W(3)BUL, a mutant lacking detectable plastids and plastid DNA, when separated on acrylamide gels, show a single band which reduces methyl viologen with NADPH, and its mobility is similar in wild type and in mutant W(3)BUL. The reductase is inducible by light and increases 3-fold in wild type in white or red light and 1.5-fold in W(3)BUL in white light. DCMU does not block chloramphenicol reduction in vivo indicating that electrons originate from sources other than photosynthetic electron transport. We infer that chloramphenicol is reduced by ferredoxin which receives electrons via ferredoxin-NADP reductase. The limiting step is not the enzyme but the source of reducing power which can be supplied from the cytoplasm, probably under control of the blue light receptor. Ferredoxin and ferredoxin NADP reductase appear to be coded in the nuclear genome, synthesized on cytoplasmic ribosomes, and join a group of enzymes which cannot be precisely localized, since they may be active anywhere from their site of synthesis in the cytoplasm to their place of deposition in the chloroplast.     

Nitrite reduction in Veillonella alcalescens.

Nitrite reduction was examined in Veillonella alcalescens C-1, and obligate anaerobe with an ATP-yielding nitrate-reducing system. Hydrogen donors for nitrite reduction included hydrosulfite, hydrogen gas, and pyruvate, but not pyridine nucleotides, in the presnece or absence of flavins. Pyruvate-linked nitrite reduction was not inhibited by 4,4,4-trifluoro-1-(2-thienyl) 1,3-butanedione, dicoumarol, or 2-heptyl-4-hydroxy-quinoline-N-oxide. The noninvolvement of membrane-bound factors was supported by the fact that 100% of pyruvate-linked activity remained in the soluble fraction after fractionation of crude extracts by ultracentrifugation. Using DEAE-cellulose column chromatography, however, the participation of ferredoxin in nitrite reduction was demonstrated. The product of nitrite reduction appeared to be ammonia, as determined from H2-to-NO2- ratios. Nitrite reductase was induced by nitrate or nitrite and was repressed by increased levels of reduced nitrogenous compounds.     

REDUCTION OF NITRATE AND NITRITE BY SUBCELLULAR PREPARATIONS OF ANABAENA CYLINDRICA II. REDUCTION OF NITRATE TO NITRITE

Reduction of nitrate to nitrite by particulate preparationsof Anabaena cylindrica was investigated. Preparations whichshowed high activity of nitrate reductase were obtained by sonication(preparation A) or acetone treatment (preparation B). The preparationA also showed a high activity of DPIP-ascorbate photooxidation.The nitrate reductase system accepted electrons from eitherreduced ferredoxin (preparation A & B) or NADH (preparationB), but not directly from NADPH. Ferredoxin was active whenreduced either by action of photochemical system I or by NADPHand NADP-reductase, but dithionite-reduced ferredoxin was completelyinactive. Ferredoxin could be replaced with methyl viologen,benzyl viologen and diquat. Reduced FMN and FAD could serveas electron donors, but the affinity of the reductase towardthese flavin compounds was very low. ¹ This work was supported by grants from the Ministry of Education(4093 and 95612) and from the National Institutes of Health,U.S. (GM-11300).     

Purification and properties of NADP-reductase of phototropic bacteria Thiocapsa roseopersicina]

The method of purification up to homogenous states and properties of NADP-reductase of purple bacteria Thiocapsa roseopersicina, strain BBS, are described. The molecular weight of NADP-reductase is about 47 000; it is flavoprotein consisting of two subunits. Atebrim and chloromercury bensoate inhibit the activity of NADP-reductase (34% and 33--60%, respectively). The enzyme is specific to NADPH; it catalyzes menadion-reductase reaction, diaphorase reaction of benzyl viologen reduction, oxidation of reduced benzyl viologen in the presence of NADP, reduction of ferredoxin and cytochrome c in the presence of NADPH, but it is not capable to catalyze transhydrogenase reaction.     

Nitrite reduction in barley-root plastids: Dependence on NADPH coupled with glucose-6-phosphate and 6-phosphogluconate dehydrogenases,and possible involvement of an electron carrier and a diaphorase

Plastids from roots of barley (Hordeum vulgare L.) seedlings were isolated by discontinuous Percoll-gradient centrifugation. Coinciding with the peak of nitrite reductase (NiR; EC 1.7.7.1, a marker enzyme for plastids) in the gradients was a peak of a glucose-6-phosphate (Glc6P) and NADP⁺-linked nitrite-reductase system. High activities of phosphohexose isomerase (EC 5.3.1.9) and phosphoglucomutase (EC 2.7.5.1) as well as glucose-6-phosphate dehydrogenase (Glc6PDH; EC 1.1.1.49) and 6-phosphogluconate dehydrogenase (6PGDH; EC 1.1.1.44) were also present in the isolated plastids. Thus, the plastids contained an overall electron-transport system from NADPH coupled with Glc6PDH and 6PGDH to nitrite, from which ammonium is formed stoichiometrically. However, NADPH alone did not serve as an electron donor for nitrite reduction, although NADPH with Glc6P added was effective. Benzyl and methyl viologens were enzymatically reduced by plastid extract in the presence of Glc6P+ NADP⁺. When the plastids were incubated with dithionite, nitrite reduction took place, and ammonium was formed stoichiometrically. The results indicate that both an electron carrier and a diaphorase having ferredoxin-NADP⁺ reductase activity are involved in the electron-transport system of root plastids from NADPH, coupled with Glc6PDH and 6PGDH, to nitrite.Abbreviations Cyt cytochrome - Glc6P glucose-6-phosphate - Glc6PDH glucose-6-phosphate dehydrogenase - MVH reduced methyl viologen - NiR nitrite reductase - 6PG 6-phosphogluconate - 6PGDH 6-phosphogluconate dehydrogenase     

The effect of low osmotic potential on nitrite reduction in intact spinach chloroplasts

The effect of water stress (reduced osmotic potential) on photosynthetic nitrite reduction was investigated using intact, isolated spinach (Spinacia oleracea) chloroplasts. Nitrite-dependent O₂ evolution was inhibited 39% at −29.5 bars osmotic potential, relative to a control at −11 bars. In the presence of an uncoupler of photophosphorylation this inhibition was not seen. Reduced osmotic potential did not inhibit either methyl viologen reduction or photosynthetic O₂ reduction. These results indicate that an inhibition of electron transport to ferredoxin cannot account for the observed inhibition of nitrite-dependent O₂ evolution. In vitro assay of nitrite reductase activity showed that the interaction of the enzyme with nitrite was not affected by changes in the concentrations of ions or molecules that might be caused by water stress conditions. These results indicate that the most likely site for the effect of water stress on chloroplastic nitrite reduction is the interaction of ferredoxin with nitrite reductase.     

Anaerobic hydrogenase activity in Anacystis nidulans. H2-dependent photoreduction and related reactions.

1. Anaerobic hydrogenase activity in whole cells and cell-free preparations of H2-induced Anacystis was studied both manometrically and spectrophotometrically in presence of physiological and artificial electron acceptors. 2. Up to 90% of the activity measured in crude extracts were recovered in the chlorophyll-containing membrane fraction after centrifugation (144 000 X g, 3 h). 3. Reduction of methyl viologen, diquat, ferredoxin, nitrite and NADP by the membranes was light dependent while oxidants of more positive redox potential were reduced also in the dark. 4. Evolution of H2 by the membranes was obtained with dithionite and with reduced methyl viologen; the reaction was stimulated by detergents. 5. Both uptake and evolution of H2 were sensitive to O2, CO, and thiolblocking agents. The H2-dependent reductions were inhibited also by the plastoquinone antagonist dibromothymoquinone, while the ferredoxin inhibitor disalicylidenepropanediamine affected the photoreduction of nitrite and NADP only. 3-(3,4-Dichlorophenyl)-1,1-dimethylurea did not inhibit any one of the H2-dependent reactions. 6. The results present evidence for a membrane-bound 'photoreduction' hydrogenase in H2-induced Anacystis. The enzyme apparently initiates a light-driven electron flow from H2 to various low-potential acceptors including endogenous ferredoxin.     

Reduction of Nitrate and Nitrite in Lambsquarters (Chenopodium album) Biotypes Resistant and Susceptible to Atrazine Toxicity

The nitrite-reducing activity of the normal susceptible biotype of lambsquarters (Chenopodium album L.) was strongly inhibited by atrazine in the assay medium, both in the case of the in vivo assays of leaf discs in light, and in vitro photoreduction assays of crude extracts. In vitro assays of crude extracts with methylviologen or ferredoxin supplying the reducing potential were not inhibited by atrazine. In the resistant biotype, inhibition of nitrite reduction did not occur with any of the above assays. Thus, it appears that atrazine does not inhibit nitrite reductase itself, but rather the availability of photosynthetically supplied electrons for the reduction. Atrazine had no effect when added to the media for either in vivo or in vitro assays of nitrate reduction by either the susceptible or resistant biotype.     

Purification and properties of nitrite reductase from the blue-green alga Anabaena cylindrical

Nitrite reductase was purified about 40-fold from the blue-greenalga Anabaena cylindrica by acetone precipitation and chromatographyon DEAE-cellulose columns. The nitrite reductase had its pHoptima at about 7.6 with Tris-HCl and at about 7.4 with phosphatewhen reduced methyl viologen was used as an electron donor.The Km's for nitrite, methyl viologen and ferredoxin were 510^–55,210^–4 and 510^–6M, respectively. A stoichiometryof one molecule of ammonia formation per one molecule of nitritedisappearance was confirmed. Ferredoxin which had been reducedeither chemically with dithionite or enzymatically with NADPHin the presence of diaphorase was active as an electron donor.Dithionite-reduced FAD and FMN were inactive. NADPH could notgive electrons directly to nitrite reductase. Hydroxylaminereductase was segregated from nitrite reductase by DEAE-cellulosecolumn chromatography. Purified nitrite reductase showed noactivity for sulfite reduction. A molecular weight of 68,000was estimated for nitrite reductase using a calibrated SephadexG-200 column. ¹This work was supported by grants 4090 and 955008 from theMinistry of Education. ²This work was supported by grants 4090 and 955008 from theMinistry of Education. 2 Present address: Department of Botany,Faculty of Science, University of Tokyo, Tokyo.     

Antigenic similarities between ferredoxin-dependent nitrite reductase and glutamate synthase fromChlamydomonas reinhardtii

Polyclonal antisera were prepared against ferredoxin-nitrite reductase (EC 1.7.7.1) and ferredoxin-glutamate synthase (glutamate synthase (ferredoxin); EC 1.4.7.1) from the green algaChlamydomonas reinhardtii. The anti-glutamate synthase antibodies recognized both glutamate synthase and nitrite reductase, but inhibited only the ferredoxin-linked activity of the latter enzyme and not the activity dependent on methyl viologen. Analogously, the anti-nitrite reductase antibodies recognized glutamate synthase and nitrite reductase but the first enzyme was only poorly inhibited. Free ferredoxin protected the nitrite reductase against its inactivation by anti-glutamate synthase antibodies. These results indicate that the ferredoxin-dependent glutamate synthase and nitrite reductase from this alga share common antigenic determinants, and that these are located at the ferrodoxin-binding domains.     

Purification and some properties of the nitrite reductase from the cyanobacterium Phormidium laminosum

Assimilatory ferredoxin-nitrite reductase (EC 1.7.7.1, ammonia: ferredoxin oxidoreductase) has been purified 5300-fold with a specific activity of 625 units/mg protein from the filamentous non-heterocystous cyanobacterium Phormidium laminosum. The enzyme was soluble and consisted of a single polypeptidic chain of 54 kDa. It catalyzed the reduction of nitrite to ammonia using ferredoxin or flavodoxin as electron donor. Methyl and benzyl viologens were also effective as electron donors but neither flavins nor NAD(P)H were. The apparent Michaelis constants for nitrite, ferredoxin and methyl viologen were 40, 22 and 215 microM, respectively. Nitrite reductase activity was inhibited effectively by cyanide and thiol reagents. The enzyme exhibited absorption maxima at 281, 391 (Soret), 570 (alpha) and 695 nm, with epsilon 391 of 4.3 x 10(4) M-1 cm-1, and an absorbance ratio A281/A391 of 1.95, suggesting the presence of siroheme as prosthetic group. These results show that this enzyme is similar to those of eukaryotic organisms.     

The reduction of nitrate, nitrite and hydroxylamine to ammonia by enzymes from Cucurbita pepo L. in the presence of reduced benzyl viologen as electron donor

1. Enzyme systems from Cucurbita pepo have been shown to catalyse the reduction of nitrite and hydroxylamine to ammonia in yields about 90–100%. 2. Reduced benzyl viologen serves as an efficient electron donor for both systems. Activity of the nitrite-reductase system is directly related to degree of dye reduction when expressed in terms of the function for oxidation–reduction potentials, but appears to decrease to negligible activity below about 9% dye reduction. 3. NADH and NADPH alone produce negligible nitrite loss, but NADPH can be linked to an endogenous diaphorase system to reduce nitrite to ammonia in the presence of catalytic amounts of benzyl viologen. 4. The NADH– or NADPH–nitrate-reductase system that is also present can accept electrons from reduced benzyl viologen, but shows relationships opposite to that for the nitrite-reductase system with regard to effect of degree of dye reduction on activity. The product of nitrate reduction may be nitrite alone, or nitrite and ammonia, or ammonia alone, according only to the degree of dye reduction. 5. The relative activities of nitrite-reductase and hydroxylamine-reductase systems show different relationships with degree of dye reduction and may become reversed in magnitude when effects of degree of dye reduction are tested over a suitable range. 6. Nitrite severely inhibits the rate of reduction of hydroxylamine without affecting the yield of ammonia as a percentage of total substrate loss, but hydroxylamine has a negligible effect on the activity of the nitrite-reductase system. 7. The apparent K_m for nitrite (1 μm) is substantially less than that for hydroxylamine, for which variable values between 0·05 and 0·9mm (mean 0·51 mm) have been observed. 8. The apparent K_m values for reduced benzyl viologen differ for the nitrite-reductase and hydroxylamine-reductase systems: 60 and 7·5 μm respectively. 9. It is concluded that free hydroxylamine may not be an intermediate in the reduction of nitrite to ammonia by plants, and a possible mechanism for reduction of both compounds by the same enzyme system is discussed in the light of current ideas relating to other organisms.     

Heparin inhibition of ferredoxin-NADP reductase in chloroplast thylakoid membranes

Heparin, an anionic polysaccharide, inhibited the ferredoxin-catalyzed reduction of NADP in spinach chloroplast thylakoid membranes. Under the same conditions of assay, heparin did not interfere markedly with photoreduction of methyl viologen, anthraquinone sulfonate, or ferredoxin. A kinetic analysis of the heparin-induced interference with NADP photoreduction showed partial competitive inhibition. Heparin also interfered with NADPH oxidation by membrane-bound ferredoxin-NADP reductase (with dichlorophenol-indophenol as the acceptor) by a mechanism that involves partial competitive inhibition. This reaction was sensitive to the presence of salts; increasing ionic strength increases the heparin Ki for inhibition of NADPH oxidation. These results show that heparin binds to ferredoxin-NADP reductase, and in doing so interferes with binding to the reductase by both ferredoxin and NADP(H). Since heparin is redox inactive and does not interfere with the photophosphorylation reaction, it is a useful inhibitor of thylakoid membrane reactions which require the catalytic activity of ferredoxin-NADP reductase.     

Studies on nitrite reductase in barley

Summary Nitrite reductase from barley seedlings was purified 50–60 fold by ammonium sulphate precipitation and gel filtration. No differences were established in the characteristics of nitrite reductases isolated in this way from either leaf or root tissues. The root enzyme accepted electrons from reduced methyl viologen, ferredoxin, or an unidentified endogenous cofactor. Enzyme activity in both tissues was markedly increased by growth on nitrate. This activity was not associated with sulphite reductase activity. Microbial contamination could not account for the presence of nitrite reductase activity in roots. Nitrite reductase assayed in vitro with reduced methyl viologen as the electron donor was inhibited by 2,4-dinitrophenol but not by arsenate.Abbreviations DNP 2,4-dinitrophenol - DEAE diethyl amino ethyl     

Complex formation between ferredoxin and Synechococcus ferredoxin: nitrate oxidoreductase

The ferredoxin-dependent nitrate reductase from the cyanobacterium Synechococcus sp. PCC 7942 has been shown to form a high-affinity complex with ferredoxin at low ionic strength. This complex, detected by changes in both the absorbance and circular dichroism (CD) spectra, did not form at high ionic strength. When reduced ferredoxin served as the electron donor for the reduction of nitrate to nitrite, the activity of the enzyme declined markedly as the ionic strength increased. In contrast, the activity of the enzyme with reduced methyl viologen (a non-physiological electron donor) was independent of ionic strength. These results suggest that an electrostatically stabilized complex between Synechococcus nitrate reductase and ferredoxin plays an important role in the mechanism of nitrate reduction catalyzed by this enzyme. Treatment of Synechococcus nitrate reductase with either an arginine-modifying reagent or a lysine-modifying reagent inhibited the ferredoxin-dependent activity of the enzyme but did not affect the methyl viologen-dependent activity. Treatment with these reagents also resulted in a large decrease in the affinity of the enzyme for ferredoxin. Formation of a nitrate reductase complex with ferredoxin prior to treatment with either reagent protected the enzyme against loss of ferredoxin-dependent activity. These results suggest that lysine and arginine residues are present at the ferredoxin-binding site of Synechococcus nitrate reductase. Results of experiments using site-specific, charge reversal variants of the ferredoxin from the cyanobacterium Anabaena sp. PCC 7119 as an electron donor to nitrate reductase were consistent with a role for negatively charged residues on ferredoxin in the interaction with Synechococcus nitrate reductase.     

The pyruvate: ferredoxin oxidoreductase in heterocysts of the cyanobacterium 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 oxidoreductase has been unambiguously demonstrated in extracts from heterocysts by the formation of acetylcoenzyme A, CO2 and reduced methyl viologen (ferredoxin) 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. alpha-Ketoglutarate: ferredoxin oxidoreductase is absent in Anabaena cylindrica, confirming that the organism has an incomplete tricarboxylic acid cycle.