Electron donation to nitrogenase in heterocysts of cyanobacteria

Various electron donors were found to stimulate C₂H₂ reduction (N₂ fixation) by isolated heterocysts from Anabaena variabilis and Anabaena cylindrica. Intermediates of glycolysis and the tricarboxylic acid cycle as well as unphosphorylated sugars like glucose, fructose and erythrose were among these electron donors. The transfer of electrons from donors like H₂, NADH, glyoxylate and glycollate was strictly light-dependent, whereas others like NADPH or pyruvate plus coenzyme A supported C₂H₂ reduction also in the dark. In all cases, the overall activity was enhanced by light. The stimulation by light was more distinct with heterocysts from A. variabilis than with heterocysts from A. cylindrica.The present communication establishes that pyruvate supports C₂H₂ reduction by heterocysts from either A. variabilis or A. cylindrica with rates comparable to those with other electron donors. Pyruvate could, however, support C₂H₂ reduction only in the presence of coenzyme A, and the concentrations of both coenzyme A and pyruvate were crucial. A pyruvate-dependent reduction of ferredoxin by extracts from heterocysts was recorded spectrophotometrically. Glyoxylate, which is an inhibitor of thiamine pyrophosphate-dependent decarboxylations, inhibited pyruvate-dependent C₂H₂ reduction. This result supports the conclusion that pyruvate is metabolised by pyruvate: ferredoxin oxidoreductase in heterocysts. High concentrations of pyruvate and other electron donors inhibited C₂H₂ reduction which suggests that nitrogenase activity in heterocysts may be controlled by the availability of electron donors.Dedicated to Professor Norbert Pfennig, Konstanz, on the occasion of his 60th birthday     

Properties and function of the pyruvate: Ferredoxin oxidoreductase from the blue-green algaAnabaena cylindrica

Extracts from the nitrogen fixing blue-green algaAnabaena cylindrica catalyse a pyruvate decarboxylation, which is dependent on ferredoxin and stimulated by coenzyme A, ATP and a SH-protecting compound. This pyruvate clastic reaction is completely reversible: The net synthesis of pyruvate requires CO₂, acetyl-coenzyme A and reduced ferredoxin. Preparations fromAnabaena cylindrica also catalyse the exchange reaction between CO₂ and the carboxyl group of pyruvate. Thus the enzyme fromAnabaena cylindrica has essentially all the characteristics known for the pyruvate: ferredoxin oxidoreductase from anaerobic bacteria.The activity of the pyruvate: ferredoxin oxidoreductase inAnabaena grown with ammonia is lower than one-fifth of that in cells grown with molecular nitrogen or nitrate as the nitrogen source. From this, it will be concluded that a physiological role of the reaction is to generate reduced ferredoxin for the assimilation of nitrogen to ammonia. The pyruvate synthesis is probably not physiological inA. cylindrica.In addition, extracts fromA. cylindrica also catalyse a ferredoxin dependent decarboxylation of -ketoglutarate. It is not yet clear, whether this ketoglutarate cleavage has a function inA. cylindrica.     

In vivo and in vitro studies on ATP and electron donors to nitrogenase in Rhodospirillum rubrum

The photosynthetic bacterium, Rhodospirillum rubrum (ATCC 11170), was tested for its ability to fix nitrogen (acetylene reduction) under aerobic and dark-anaerobic conditions. Whole cells reduced acetylene under darkanaerobic conditions if pyruvate was supplied. Reactions of the cells were inhibited less by oxygen in the dark than in the light, and the cells were capable of acetylene reduction in the presence of low levels of oxygen (0.6%) in the dark. Crude extracts of R. rubrum reduced acetylene if pyruvate and Coenzyme A were added; ferredoxin from R. rubrum greatly increased the pyruvate-driven activity in crude extracts. It was not possible to demonstrate light-driven acetylene reduction in crude extracts unless a reductant (dithionite) was added.Abbreviations Fld flavodoxin - DTT dithiothreitol     

The effect of pyruvate on nitrogenase activity in the blue-green alga Anabaena cyclindrica

Exogenous pyruvate added to cultures of the bluegreen alga, Anabaena cylindrica stimulated nitrogenase activity (measured by acetylene reduction) only in the dark under low pO₂ (0.05 atmospheres). Under aerobic conditions or in the light, stimulation was absent and replaced by an inhibition of activity above 5 mM added pyruvate. The curve of nitrogenase activity versus oxygen concentration had a similar maximal value of ethylene production with or without added pyruvate, but in the presence of pyruvate this maximum occurred at 0.05 atmospheres O₂, whilst in the absence of pyruvate the maximum occurred at 0.10 atmospheres O₂. Malate, citrate, α-ketoglutarate, glucose and fructose were tested also, but none gave a similar effect to pyruvate. Addition of ¹⁴C-pyruvate and autoradiography indicated that exogenous pyruvate is metabolized through the interrupted Krebs cycle. These results are explained in terms of the activity of pyruvate: ferredoxin oxidoreductase and the ATP-induced oxygen sensitivity of nitrogenase.     

Pyruvate-supported acetylene and sulfate reduction by cell-free extracts of Desulfovibrio desulfricans

Cell-free extracts prepared from a strain of $\text{Desulfovibrio}\text{desulfricans}$ can reduce acetylene or sulfate, utilizing pyruvate as the electron and ATP source in the presence of methyl viologen, ADP and coenzyme A. Other physiological substances such as (lactate + NAD) and (NADH + ATP) can not reduce acetylene nor sulfate. When acetylene and sulfate are both present as substrates, sulfate represses the acetylene reduction.     

Hydrogenase activity in the thermophile Mastigocladus laminosus

Hydrogenase activity in the thermophilic cyanobacterium, $\text{Mastigocladus}$$\text{laminosus}$ was studied both $\text{in vivo}$ and $\text{in vivo}$ hydrogen consumption required oxygen but not light, was about ten-fold higher than in mesophilic cyanobacteria, and was relatively insensitive to carbon monoxide. H₂-supported acetylene reduction in reductant-limited cultures was a light-dependent, but O₂-independent reaction. $\text{In vitro}$ hydrogen evolution was unaffected by carbon monoxide, and this activity could be partially purified using a procedure developed for $\text{Anabaena cylindrica}$.     

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.     

Modes of reduction of nitrogenase in heterocysts isolated from Anabaena species

N₂ fixation (acetylene reduction) has been studied with heterocysts isolated from Anabaena cylindrica and Anabaena 7120. In the presence of ATP and at very low concentrations of sodium dithionite, reducing equivalents for activity of nitrogenase in these cells can be derived from several compounds. In the dark, d-glucose 6-phosphate, 6-phosphogluconate and dl-isocitrate support acetylene reduction via NADPH. In the light, reductant can be generated by Photosystem I.     

A constitutive flavodoxin from a eukaryotic alga

We report the isolation and some properties of a flavodoxin from a eukaryotic organism, the naturally occurring red alga $\text{Chondrus crispus}$. Unlike the situation with most other organisms the flavodoxin, under normal growth conditions, is the predominantly formed low-potential electron carrier, an accompanying ferredoxin occurring in only very small amounts. The flavodoxin is of molecular weight 21000 and one mole of FMN is present per mole of protein. Reduction of the flavoprotein proceeds via a blue flavosemiquinone radical. The flavodoxin is active both in photosynthetic NADP reduction by broken chloroplasts, and in phosphoroclastic cleavage of pyruvate by cell-free extracts of $\text{Clostridium pasteurianum}$.     

Chemical modification of spinach ferredoxin: evidence for the involvement of a complex between ferredoxin and ferredoxin:NADP oxidoreductase in NADP photoreduction.

Spinach ferredoxin was modified chemically with trinitrobenzene sulfonic acid (TNBS), a reagent which reacts specifically with amino groups. The trinitrophenylated ferredoxin (TNP-Fd) can accept electrons from Photosystem I as indicated by its full activity in the photoreduction of cytochrome $\text{c}$. The modified protein is inactive, however, in the photoreduction of NADP and cannot form a complex with the flavoprotein, ferredoxin: NADP oxidoreductase. The data presented indicate that the inactivity of the modified protein is the result of modification of a single amino group.     

Identification of carboxylation enzymes and characterization of a novel four-subunit pyruvate:flavodoxin oxidoreductase from Helicobacter pylori.

The enzyme activities responsible for carboxylation reactions in cell extracts of the gastric pathogen Helicobacter pylori have been studied by H14CO3- fixation and spectrophotometric assays. Acetyl coenzyme A carboxylase (EC 6.4.1.2) and malic enzyme (EC 1.1.1.40) activities were detected, whereas pyruvate carboxylase (EC 6.4.1.1), phosphoenolpyruvate carboxylase (EC 4.1.3.1) and phosphoenolpyruvate carboxykinase (EC 4.1.1.49) activities were absent. However, a pyruvate-dependent, ATP-independent, and avidin-insensitive H14CO3- fixation activity, which was shown to be due to the isotope exchange reaction of pyruvate:flavodoxin oxidoreductase (EC 1.2.7.1), was present. The purified enzyme is composed of four subunits of 47, 36, 24, and 14 kDa. N-terminal sequence analysis showed that this enzyme is related to a recently recognized group of four-subunit pyruvate:ferredoxin oxidoreductases previously known only from hyperthermophiles. This enzyme from H. pylori was found to mediate the reduction of a number of artificial electron acceptors in addition to a flavodoxin isolated from H. pylori extracts, which is likely to be the in vivo electron acceptor. Indirect evidence that the enzyme is capable of in vitro reduction of the anti-H. pylori drug metronidazole was also obtained.     

Pyruvate oxidation by the Reiter strain of Treponema phagedenis.

Spectrophotometric assays of pyruvate oxidation catalyzed by extracts of the Reiter strain of Treponema phagedenis indicated that viologen dyes, flavin nucleotides, and a ferric iron chelate, but not pyridine nucleotides, were utilized as electron acceptors. Benzyl viologen-linked activity partially sedimented during ultracentrifugation and appeared similar to clostridial pyruvate:ferredoxin oxidoreductase with respect to the spectral properties of the enzyme chromophore. Electron carrier activity in treponemal extracts was quantitated by a metronidazole-linked assay in which the oxidation of pyruvate by carrier-depleted extracts led to the reduction of electron carrier in the crude extracts which then reduced metronidazole. The rate of metronidazole reduction was proportional to the amount of electron carrier present in the assay. Electron carrier activity in Triton X-100-solubilized, crude extracts partially purified by DEAE-cellulose chromatography and gel filtration was attributed to a protein possessing the spectral and physical properties of a ferredoxin. A similar protein appeared to be present in extracts of Treponema denticola ST10.     

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.     

The defect in the cblB class of human methylmalonic acidemia: Deficiency of cob(I)alamin adenosyltransferase activity in extracts of cultured fibroblasts

We show that the reductants present in the $\text{in}\text{vitro}$ assay used to measure the formation of adenosylcobalamin from cob(III)alamin by cell-free extracts of human fibroblasts result in the non-enzymatic reduction of cob(III)alamin to cob(I)alamin. Hence, the $\text{in}\text{vitro}$ assay uniquely estimates the activity of ATP:cob(I)alamin adenosyltransferase (EC 2.5.1.17). Based on additional studies with extracts of fibroblasts from patients in the $\text{cbl}\text{B}$ class of human methylmalonic acidemia and from their parents, we conclude that this mutant class results from a specific deficiency of adenosyltransferase activity which is inherited as an autosomal recessive trait.     

Pyruvate: Ferredoxin oxidoreductase and its activation by ATP in the blue-green alga Anabaena variabilis

1. The breakdown of pyruvate was examined in whole cells and cell-free extracts of the blue-green alga Anabaena variabilis. Decarboxylation of specifically labelled pyruvate indicated a similar metabolic route to that of acetate, although no pyruvate oxidase was present. Pyruvate: ferredoxin oxidoreductase was detected in cell-free extracts and after DEAE-cellulose treatment, addition of ferredoxin was necessary for pyruvate decarboxylation; acetyl-CoA was the first product of the reaction.     

Separation of L-phenylalanine: pyruvate transaminase and L-phenylalanine: alpha-ketoglutarate transaminase by sephadex-electrophoresis.

By means of a Sephadex-electrophoresis column, L-phenylalanine: pyruvate transaminase (PPT) was separated from L-phenylalanine: α-ketoglutarate transaminase (PKT) from rat liver. These enzymes differed in heat lability $\text{in vitro}$ and in their inducibility by glucagon $\text{in vivo}$. PPT was heat-stable and was induced by chronic glucagon injection. On the other hand, PKT was heat-labile and was not induced by glucagon under the experimental conditions used. These studies provide evidence that distinct enzymes catalyze the transamination of phenylalanine with pyruvate or with α-ketoglutarate as the amino acceptor.     

Pyruvate dehydrogenase complex,pyruvate: Ferredoxin oxidoreductase and lipoic acid content in microorganisms

Several microorganisms were examined for the content of lipoic acid by using a strain of Streptococcus faecalis deficient in this coenzyme. In comparison to this, the specific activity levels were determined for the pyruvate: ferredoxin oxidoreductase and the pyruvate dehydrogenase complex, which both catalyse the cleavage of pyruvate and coenzyme A to acetyl coenzyme A, CO₂ and two reducing equivalents. Anabaena cylindrica, Chlorobium, Clostridium pasteurianum and kluyveri, where only the pyruvate: ferredoxin oxidoreductase can be demonstrated, were found to contain minute levels of lipoic acid. Thus lipoic acid does not appear to be a cofactor of the decarboxylation catalysed by the pyruvate: ferredoxin oxidoreductase. On the other hand, the amount of lipoic acid is at least ten times higher in Ankistrodesmus, Chlamydomonas, Anacystis, Micrococcus, Azotobacter and Escherichia coli which have the dehydrogenase complex.     

Effect of carbamyl phosphate on the regulation of nitrogenase in Clostridium pasteurianum

One mM carbamyl phosphate inhibited the $\text{in}$$\text{vitro}$ acetylene reduction activity of nitrogenase 30% whereas at high concentrations a maximum inhibition of 50% was observed. When 1 mM carbamyl phosphate was added to a culture growing of N₂ 1) nitrogenase synthesis was completely repressed and 2) after a period of 2.5 hrs in the absence of growth, the specific activity decreased to less than 50% of its activity just before the addition of the inhibitor.     

Utilization of sodium-dependent high affinity choline uptake in vitro as a measure of the activity of cholinergic neurons in vivo.

Previous reports indicate that alterations of activity of cholinergic neurons $\text{in vivo}$ are followed by parallel changes in sodium-dependent high affinity choline uptake $\text{in vitro}$. These results are consistent with the proposal that this portion of choline uptake is regulatory in the synthesis of ACh. These results also suggest the possibility of utilizing sodium-dependent high affinity choline uptake as a measure of the relative state of cholinergic activity $\text{in vivo}$. In this study, we administer a number of drugs reported to alter turnover and release of ACh (both are measures of cholinergic activity $\text{in vivo}$, and subsequently examine sodium-dependent high affinity choline uptake $\text{in vitro}$. Administration of pentobarbital, chloral hydrate, morphine, physostigmine, Δ⁹ THC, hemicholinium-3 and oxotremorine, drugs which decrease ACh turnover and release, caused a reduction in choline uptake. Conversely, administration of pentylenetetrazol, atropine, scopolamine, and haloperidol, drugs which increase ACh turnover and release, caused an increase in choline uptake $\text{in vitro}$. These findings support the proposal that sodium-dependent high affinity choline uptake can be used as a relative measure of the activity of cholinergic neurons $\text{in vivo}$.