6 research outputs found
Aspects of the oxygen-tolerance of nitrogen fixation in Azotobacter vinelandii
No full textFollowing the pioneering physiological studies of Haaker et al. on the generation of reducing equivalents for nitrogenase in Azotobactervinelandii, it seemed desirable to further characterize interactions between the likely ultimate electron donors (flavodoxin and/or ferredoxin) and nitrogenase. Isolation of these proteins was therefore necessary. Flavo- and ferredoxin from A.vinelandii have been prepared to purity; as far as nitro genase was concerned, however, it was deemed that the O 2 -tolerant nitrogenase complex as isolated originally by Bulen and LeComte should render information more physiologically relevant than could be the case using highly puri fied nitrogenase components (Av 1 + Av 2 ). In chapter II, results are described suggesting that the Bulen-LeComte nitrogenase complex, with fully reduced fla vodoxin as a source of reducing equivalents, has regulatory properties not exhibited by a more highly purified (Av 1 + Av 2 ) complex, due to the presence of a 'contaminating' third protein.This third protein appeared to be the same as that shown by Haaker etal. to be responsible for the oxygen-tolerance of the nitrogenase complex. The spectral properties and molecular weight are shown in chapter II to be identical to that of the [2Fe-2S] ferredoxin isolated earlier by Shethna etal. who, however, did not ascribe any physiological function to this pro tein. In chapters III and IV, the interactions between Av 1 , Av 2 and the third protein are further characterized, as well as the conditions necessary to induce the reconstitution of an oxygen-tolerant complex from the purified proteins. The relation between the oxygen-tolerant complex and the so-called 'switched-off state' of nitrogenase activity in vivo is discussed
Aerobic nitrogen fixation in Azotobacter vinelandii
No full textI ELECTRON DONATION TO NITROGENASEPaper I shows that the hypothesis, that a high ratio of (NADH + NADPH) / (NAD + + NADP + ) is the source of reducing power for nitrogenase in intact A.vinelandii, is invalid. On the contrary, with a decreasing ratio of reduced to oxidized pyridine nucleotides, the nitrogenase activity of whole cells increases. The experiments described in paper I, indicate that the reducing power necessary for nitrogen fixation in A.vinelandii is generated within the cytoplasmic membrane. It is demonstrated that transport of reducing equivalents to the nitrogenase requires a high energy level of the cytoplasmic membrane. The energy level of the cytoplasmic membrane was measured by the intracellular Alp concentration and by using 9-amino acridine as a fluorescent probe. Other regulating factors of the nitrogenase activity in A.vinelandii are shown to be the intracellular ATP/ ADP ratio and the presence of oxygen.Paper III shows that toluene makes A.vinelandii cells permeable for small molecules but not for enzymes. In toluene-treated cells, enzyme activities can be measured by adding the appropriate cofactors and substrates. It is possible to restore the oxidation of organic substrates but no concomitant nitrogenase activity can be observed. We suggest that an observed lack of energization of the cytoplasmic membranes is the missing link between oxidation and generation of the reducing equivalents for nitrogen fixation. In paper III and IV we show that the endogenous low potential electron carriers in toluene-treated cells are not reduced. In paper IV a membrane-bound NAD(P)H-flavodoxin oxidoreductase is demonstrated and a proposal is given in which NADH is the electron donor for nitrogenase. The electron carrier flavodoxin is reduced by the membrane-bound NADH-flavodoxin oxidoreductase at a low pH, that is developed in an energy-linked process.II OXYGEN PROTECTION OF NITROGENASEIn paper II the source of respiration protection is investigated. Experiments with radioactive pyruvate and sucrose show that the rate of sucrose oxidation by A.vinelandii is associated with the sucrose translocator activity. We show that the respiration protection of the nitrogen-fixing system in A.vinelandii is dependent of the oxygen input during growth. The oxidation capacity intrinsically depends on the type of substrate and can be partly adapted.Membranes rich in cytochromes c 4 + c 5 and o and with phosphorylation between NADH and c 4 + c 5 and oxygen and cytochrome c 4 + c 5 and oxygen, can be isolated from A.vinelandii grown O 2 -limited. Cytochromes b and d can be detected in addition when A.vinelandii cells are grown N 2 limited. The activity of the NADH oxidase system is increased in such cells and phosphorylation is only observed between CoQ and oxygen. Under saturating oxygen concentrations the type of respiratory membranes was not observed to influence the intracellular energy charge.In paper III and IV the mechanism of the conformational protection of nitrogenase was investigated. It is shown that nitrogenase can be isolated as an oxygen-stable complex form A.vinelandii independent of the cell rupture method. Also no influence of the cell rupture method on the rate of sedimentation of the nitrogenase can be observed. The rate of sedimentation of the nitrogenase is found to be concentration and pH dependent. At pH=7.4 the rate of sedimentation of the nitrogenase complex is comparable with that of the pyruvate dehydrogenase complex.No evidence was found for a particulate nitrogenase, it is demonstrated that the oxygen stability of nitrogenase in crude extracts is caused by complexation. of the nitrogenase components with an Fe-S protein. An alternative proposal for the switch-on switch-off phenomenon in whole Azotobacter cells is given. Nitrogenase is present in vivo as an active and oxygen tolerent complex but nitrogen fixation in whole cells is inhibited by the oxidation of flavodoxin hydroquinone.<p/
The involvement of the fixABCX genes and the respiratory chain in the electron transport to nitrogenase in Azotobacter vinelandii
No full textIntroduction.The work in this thesis is mainly focused on the electron transport route to nitrogenase in the free-living, obligate aerobic, nitrogen fixing organism Azotobacter vinelandii. For many years now, this topic has been the subject of research. Several hypotheses, which would explain the mechanism of electron transport to nitrogenase in obligate aerobic bacteria, have been postulated. None of these hypotheses have been proven yet.The electron transport to nitrogenase in A.vinelandii has been investigated both biochemically and genetically. It is known in Klebsiella pneumoniae, which fixes nitrogen anaerobically or microaerobically, that the gene products of two genes are responsible for the electron transport to nitrogenase, the nifF gene and the nifJ gene. They encode a flavodoxin and a pyruvate: flavodoxin oxidoreductase, respectively. Electrons are transferred from pyruvate to flavodoxin through this oxidoreductase, and are then passed on to the nitrogenase proteins. This reaction, nor the two genes involved, have been found in A.vinelandii. There must therefore be different pathway for electrons to reduce nitrogenase in this organism.Gubler and Hennecke [1986] discovered a number of genes, the fixA , B and C genes, which were required for nitrogen fixation in the obligate aerobic Bradyrhizobium japonicum, both in symbiotic and free-living state. Later, they found that another gene, linked to the fixBC cluster, the fixX gene, was also involved in nitrogen fixation. Since the nodules of plants, infected with mutants in these genes were normal, but no nitrogenase activity was observed, a function in the electron transport to nitrogenase was suggested. This was contradicted later by Kaminski and coworkers [1989], who found that both in vivo and in vitro nitrogenase activity was absent in mutants in the fixABCX genes in Azorhizobium caulinodans ORS571.The fixPABCX genes of A.vinelandii: genetic analysis.In order to find out whether these genes are also present in A.vinelandii, a 4.4 kb part of the genome of this organism, which hybridised to a heterologous fixA probe from Rhizobium leguminosarum was isolated. The nucleotide sequence of the 4.4 kb Sma I -Eco RI fragment of Azotobacter vinelandii was determined. Five open reading frames (ORF's) and the beginning of a sixth one were found. The proteins encoded by the open reading frames were investigated for their homologies with other known gene products in the Genbank ®databank.The nucleic acid derived protein sequence of the first open reading frame contains two cysteine patterns, [Cys-X7-Cys-X3-Cys] and [Cys-X2-Cys-X2-Cys-X3-Cys], indicative for the ligation of both a [3Fe-4S] and a [4Fe-4S] and a [4Fe-4S cluster. These sequences are characteristic for 7Fe-ferredoxins, such as ferredoxin I of A.vinelandii (FdI). Besides the conserved cysteine residues, no other regions of homology between the fixP gene product and ferredoxin I were found, and no apparent homology with any other protein in the database was foundThe second open reading frame showed a high degree of homology with the fixA gene product of various other aerobic nitrogen fixing bacteria. The A.vinelandii fixA gene product did not have any homology with other proteins in the database, so its function could not be determined from the nucleic acid derived protein sequence.Homology searches revealed that the product of the third open reading frame not only had a high degree of homology with the sequence of FixB proteins, but also with the protein sequence of the α-subunit of the Electron Transfer Flavoprotein (ETF) of both human and rat origin. No other significant homologies with the A. vinelandii fixB gene product were found in the database.The fourth open reading frame was homologous with Rhizobial FixC proteins. The N- terminal domain of the A. vinelandii fixC gene product was found also to contain a sequence homologous with the consensus sequence for an ADP binding site, as found in NAD +or FAD dependent enzymes. In many FAD-containing enzymes ( e.g. lipoamide dehydrogenase and mercuric reductase), the FAD-binding site is located close to the N- terminus. This suggests, that the FixC protein might be a FAD-containing protein. It is known, that the β-subunit of ETF contains FAD. However, as no primary structure of this subunit has been published yet, it is not known if FixC has homology to the β-subunit of ETF.The protein encoded by the fifth open reading frame is also homologous with ferredoxin I of A.vinelandii and also with the FixX proteins of various Rhizobia, all ferredoxin-like proteins. The A.vinelandii FixX protein is the only FixX protein, that contains the two cysteine motifs that are found in ferredoxin I and the FixP protein, whereas the FixX proteins of other nitrogen fixing bacteria lack the cysteine motif, involved in ligation of the [3Fe-4S] cluster.The fact that A.vinelandii contains at least five different 7Fe-ferredoxins (FdI, FdN (7Fe-ferredoxin in the nif gene cluster), FdV (7Fe-ferredoxin in the alternative nitrogenase gene region), FixP and FixX) is indicative for a special function of these proteins in this organism. Recently, a hypothesis has been proposed by Thomson [1991], stating that the function of the 7Fe- ferredoxins in A.vinelandii is to regulate gene expression by binding to the DNA. This binding is controlled by the iron(II) levels and the redox state of the cell. When the 7Fe-ferredoxin-DNA complex binds iron(II), it becomes an 8Fe-ferredoxin. The affinity of the 8Fe-ferredoxin for the DNA is less than that of the 7Fe ferredoxin, so the ferredoxin no longer binds to the DNA, and mRNA synthesis is possible. The fact that A.vinelandii contains five genetically distinct 7Fe-ferredoxins could be clue for this model, whereas the existence of two different 7Fe-ferredoxins in what is most likely one operon, might give reason to assume that the fixPABCX cluster is involved in regulation of some kind of process, possibly involved in nitrogen fixation.Recently, the fixABCX genes have also been found in the 0-2.4 min region of the Escherichia coli genome. E.coli is unable to grow diazotrophically. The E.coli fixABCX genes were followed by an open reading frame encoding a NAD(P)H dehydrogenase and preceded by a number of genes encoding proteins, involved in fatty acid metabolism. This might be a clue for a function of these genes in fatty acid metabolism, which could be of vital importance for nitrogen fixing organisms.Downstream of the fixX gene, the start of a sixth open reading frame was found, but the N-terminal sequence did not show any homology to other proteins in the database.A sequence motif with high homology to the promoter consensus for RNA polymerase complexed with sigma factor 54 (σ 54) was found 63 bp upstream of the ATG start codon of the fixP gene. A putative binding site for the regulatory NifA protein (TGT-N 9 -ACA) is found 164 bp upstream of the start codon. However, the spacing between the TGTand ACA- elements of this sequence is one base shorter than the consensus (TGT-N 10 -ACA). The reason for this mismatch is not known, but it is known that a mismatch like this still functions in other organisms. No terminator sequence was found downstream of the stop codon of any of the genes.It is concluded that amongst the fixPABCX genes of Azotobacter vinelandii at least three genes encode proteins, which are possibly involved in electron transport: FixB is highly homologous to the α-subunit of ETF and both FixP and FixX are homologous to A. vinelandi ferredoxin I. Whether these genes are actually involved in an electron transport system, fatty acid metabolism or whether they fulfil a function in the proposed regulation of gene expression in A.vinelandii, is the objective of the research described in chapter 3.the fixPABCX genes of A.vinelandii: physiological analysisChapter 3 describes the construction and characterisation of mutants of A.vinelandii with alterations in the fixA, fixB, fixC and/or fixX genes. The gene of interest was exchanged with a plasmid derived copy that had been interrupted by insertion of the gene encoding kanamycin-resistance. A mutant lacking the fixABCX genes was constructed by replacing these genes by a DNA fragment containing the gene encoding kanamycin resistance.The mutants were tested under a large number of conditions. All fix-mutants showed normal growth characteristics in nitrogen-free medium under all conditions tested. In vivo and in vitro activities of acetylene reduction of mutants were comparable to wildtype activities. Growth on several sugars, dicarboxylic acids and fatty acids or amino acids was not different from wild type bacteria, which indicated that the fixABCX cluster is not obligatory in the catabolism of these components.Our results indicate that there is a major difference between the fixABCX genes of various Rhizobia and those of A.vinelandii. In contrast to A.vinelandii, deletion of the genes in symbiotic nitrogen fixing organisms results in loss of both in vivo and in vitro nitrogenase activity. The finding, that in R.leguminosarum, the polypeptides from which the nitrogenase enzyme complex is composed, are present in FixA -, B -, and C -mutants but inactive, suggests that a step in the biosynthesis of active nitrogenase enzyme is hampered in these bacteria or that the proteins are inactivated by oxygen damage during growth. In A.caulinodans, a FixC -mutant still had 10% of wild type nitrogenase activity, which could be elevated to 36% by adding saturating amounts purified nitrogenase Fe protein. This is an indication that the fixC gene product is necessary for the maturation of nitrogenase of A.caulinodans. In A.vinelandii no similar function for the fixABCX genes could be demonstrated.The hypothesis of Thomson that the 7Fe-ferredoxin of Azotobacter might be a DNA binding protein, involved in regulation of protein synthesis in response to iron(II) levels in the cell, could be an indication for the function of the fixPABCX genes in A.vinelandii. The fixPABCX gene cluster contains two 7Fe-ferredoxins, but evidence for the iron(II) dependent regulation has not been found.the fixPABCX genes of A.vinelandii: promoter analysis.The expression of the fixPABCX genes was investigated using two methods. A chromosomally integrated fixA :: lacZ gene fusion was made and it was observed that expression of the fixABCX genes of A.vinelandii was not significantly increased when cells, grown in the presence of ammonium, were transferred to a nitrogen free medium. It was concluded that the expression of the fixABCX genes, if occurring, was very low. From experiments, in which the promoter activity was investigated by immunological techniques, using antibodies against the purified FixA protein, similar observations were made. A very low signal of the FixA protein on the Western blots was found. Approximately half of this signal was found in cell extracts, grown in the presence of ammonia, and even in a FixA -mutant, a weak signal was detected. This signal was probably caused by a-specific binding of the antibodies, since 300 μg of total cellular protein was loaded in one slot of the gel.It cannot be ruled out, that downstream of the fixX gene, one or more genes are located that are co-transcribed with the fixPABCX genes. A mutation in the fixPABCX genes might cause polar effects on the downstream genes. The fact however, that no effect of any of the mutations was found, is either evidence that no polar effect is present, or that the gene(s) downstream of the fixX gene is/are a negative regulatory gene(s).It is concluded, that, the fixABCX genes, which are of vital importance for nitrogen fixation in symbiotic organisms, are not essential for nitrogen fixation in A.vinelandii. Despite all investigations the function of the fixABCX genes is not known.Electron transport to nitrogenase: biochemical investigations.Biochemical investigations on the electron transport to nitrogenase are the subject of chapter 4. In order to elucidate the electron pathway to nitrogenase, a model system was used, in which the flavodoxins were replaced by artificial low potential electron carriers. The respiration and the reduction of viologens by different substrates, catalysed by Azotobacter vinelandii cytoplasmic membranes was investigated. Only with NADH, viologen oxidoreductase activity could be detected; NADPH, malate, succinate and lactate were unable to reduce viologens. From the oxygen consumption experiments with different substrate combinations it is concluded, that NADH and NADPH are oxidised by different dehydrogenases, although they have the same output site in the respiratory chain towards ubiquinone. Malate dehydrogenase on the other hand, has a different output site to ubiquinone than the dehydrogenases that oxidise NADH and NADPH.The kinetic parameters of the NADH:ubiquinone oxidoreductase of the respiratory chain were investigated and compared with the NADH:ferricyanide oxidoreductase activity, which is an activity of complex I of the respiratory chain. In contrast to the NADH:ferricyanide oxidoreductase activity, the NADH:viologen oxidoreductase activity did not show double substrate inhibition, which indicates that the viologen reducing site of the NADH dehydrogenase complex is different from the NADH binding site. EPR studies on the presence of paramagnetic centers in cytoplasmic membranes demonstrated a 1owpotential" electron accepting site, which could only be reduced using dithionite. This indicates that either the site is not accessible for NADH, or the redox potential is too low to enable NADH to reduce this site.Viologen reduction did not only take place under conditions of an inhibited respiratory chain, but also under aerobic conditions with an active respiratory chain. This shows that electrons from NADH are transferred to the viologens, when at the same time electrons from NADH are transferred through the respiratory chain to oxygen.The NADH:viologen oxidoreductase activity was modulated by using different viologens and by changing the viologen concentration. It was observed that the hydrogen peroxide formation increased linearly with the NADH:viologen oxidoreductase activity. It was also observed that maximally 50% of the electrons from NADH were transferred to the viologens and that the NADH-dehydrogenase activity (modulated by the NADH/NAD +) had no influence on the distribution over viologens and ubiquinone.The results of the experiments can be used as an example for a model, as proposed by Haaker and Klugkist [1987]. Some modifications should be made to update the model to the current knowledge. In the model, a NADPH dehydrogenase is the central part, which, according to the results of this work, should be altered to a NADH dehydrogenase, since no viologen reduction was observed when NADPH was used as electron donor. No statements can be made to whether or not the 29kDa protein is involved in the reduction of low potential mediators. The concomitant flow of electrons through the respiratory chain and electron transport to low potential redox mediators, the central dogma of the model of Haaker and Klugkist, is shown in this work. This supports the observation of Klugkist et al. [1986], that electron transport to nitrogenase and respiration are coupled. According to the revised model, shown in figure 1, two electrons from NADH are accepted by the FMN group of the NADH dehydrogenase, operating at -320mV. During respiration, these electrons are subsequently distributed over respiration and a route, leading to the reduction of viologen. In the presence of cyanide and oxygen, the electrons can only be directed to the viologen reducing cluster. The viologens are oxidised efficiently by dioxygen to form H 2 O 2 , thereby maintaining a high concentration of oxidised viologen. The reduced FMN group of the NADH dehydrogenase can also be oxidised by ferricyanide.The electron transport pathways as suggested in Figure 1 explain why viologen reduction and respiration are coupled and why not more than 50% of the electrons from NADH are used for viologen reduction. Unfortunately, the formation of H 2 O 2 was not observed when the viologens were replaced by purified flavodoxin. The fact that flavodoxin reduction is not observed under the conditions applied, could be an indicationfor the existence of a factor, which is absent under the experimental conditions. This factor could be an oxygen sensitive and/or a soluble protein. A possible candidate for an O 2 sensitive, membrane bound protein could be the 29kDa protein, found by Klugkist and coworkers. It is also possible that this factor is present in a complex of proteins, which is lost apart during the isolation of the membranes. Future research is required to investigate the possibility to link flavodoxin reduction to respiration
The symbiosis between Rhizobium leguminosarum and Pisum sativum : regulation of the nitrogenase activity
No full textBacteria of the genus Rhizobium can form a symbiosis with plants of the family Leguminosae. Both bacteria and plant show considerable biochemical and morphological changes in order to develop and carry out the symbiosis. The Rhizobia induce special structures on the legumes, which are called root nodules. In these root nodules, the differentiated bacteria - so-called bacteroids - are localized. Within the root nodule the bacteroids are able to reduce atmospheric N 2 to NH 3 , which - after assimilation - is used by the plant. In turn, the plant supplies the bacteroids with carbon compounds from which the energy required for the N 2 -reduction is derived.The N 2 -reduction within the bacteroids is catalyzed by the enzyme nitrogenase. Nitrogenase requires for activity energy in the form of ATP and a low potential electron donor. An anaerobic environment at the site of nitrogen fixation is a requirement for nitrogenase because O 2 inhibits the activity of this enzyme. However O 2 is necessary for the respiration of the bacteroids. Without bacteroid respiration, no ATP is synthesized and no reducing equivalents are generated, which are both required for nitrogenase activity. This means that the O 2 supply to the bacteroids must be strictly regulated.As a side reaction during N-reduction, H +is reduced. Consequently, by reducing H +ATP and reducing equivalents are consumed. Under optimum condition, about 75 % of the electron flow through the nitrogenase reaction is utilized for the reduction of N2. The remainder is consumed in the reduction of H +. The apparent waste of energy through H +-reduction can be much greater than 25 %. The magnitude of loss is influenced by many factors.The aim of the experiments described in this thesis, is to identify the plant factors which determine the nitrogenase activity and the electron allocation to N 2 and H+ by nitrogenase. The experiments were performed with Rhizobium leguminosarum strain PRE and the host plants Pisum sativum cv. Rondo and Pisum sativum cv. Finale. Different physiological aspects underlying the functioning of the root nodule, were studied, namely:- the role of malate dehydrogenase in the supply of oxidizable substrates to the bacteroids- the role of glutamate oxaloacetate transaminase in the NH 3 assimilation and the exchange of metabolites between the symbionts in the root nodule- the influence of the external pH of bacteroids on bacteroid respiration and nitrogen fixation- the relationships between the bacteroid respiration, the intracellular ATP/ADP ratio and nitrogenase activity.In chapter 2, the presence of root nodule-stimulated forms of malate dehydrogenase is demonstrated. From a comparison of the kinetic properties of the predominant nodule-stimulated form and the main malate dehydrogenase form from uninfected root cells, it is concluded that the nodule-stimulated form is capable of catalyzing a high rate of malate formation from oxaloacetate. The second conclusion drawn from the kinetic data is that under physiological conditions the reduction of oxaloacetate to malate catalyzed by the nodule-stimulated form is inhibited at higher malate concentrations. Only the nodulestimulated form exhibits this kinetic property. This prevents the enzyme from catalyzing the reaction to equilibrium, which would lead to a very low oxaloacetate concentration in the cytoplasm of the root nodule cells. The malate concentration has to be controlled because malate is the main substrate of the bacteroids, it plays a central role in the metabolism of the mitochondria and malate -being a strong acid - affects the pH.In chapter 3, the action of malate/aspartate shuttle between the cytoplasm of the infected plant cell and the bacteroid has been demonstrated. The involvement of a nodule-stimulated glutamate oxaloacetate transaminase, present in the cytoplasm of root nodule cells, in the shuttle is suggested. The shuttle might have the following functions for nitrogen fixation.The shuttle can transport NADH from the cytoplasm of the nodule plant cells to the bacteroid, where NADH can be oxidized by the respiratory chain. The second function of the shuttle is the transamination of oxaloacetate to aspartate in the bacteroid. The aspartate formed in the bacteroid, is transported at high rates to the cytoplasm of the nodule. This is important because labelling studies of other investigators with 14C-labelled aspartate have demonstrated that aspartate is rapidly converted to malate in the cytoplasm of nodule plant cells. The aspartate formed in the bacteroid and transported to the plant cytoplasm by the shuttle, can replenish the loss in aspartate in the plant cytoplasm. The presence of a sufficient concentration of aspartate is necessary for the asparagine synthesis, a reaction of the NH 3 assimilation.In chapter 4, the effect of O 2 on nitrogenase activity and the electron allocation by nitrogenase in the root nodules and in the bacteroids, has been described. Oxygen limitation in bacteroids results in a decreased nitrogenase activity and a decreased electron allocation to N 2 by nitrogenase. In root nodules, the O 2 limitation causes also a decrease in nitrogenase activity, however the electron allocation remains constant. It is shown that the external pH of bacteroids determines the rate of respiration by the bacteroid and consequently the rate of nitrogenase activity, without affecting the electron allocation by nitrogenase. By comparing the electron allocation by nitrogenase in root nodules and that in isolated bacteroids, it is proposed that in the intact root nodule the nitrogenase activity is modulated by the pH.In chapter 5, the mechanism is studied by which the external pH of bacteroids changes the rate of respiration and the rate of nitrogenase activity at low O 2 concentrations. The relationships between the rate of respiration by the bacteroid, the nitrogenase activity and the intracellular ATP/ADP ratio are determined.The results demonstrate that a high rate of respiration of the bacteroids at low free O 2 concentrations is associated with an intracellular ATP/ADP ratio which is lower than ≈1.2 . A high rate of respiration is necessary to achieve maximum nitrogenase activity. When the intracellular ATP/ADP ratio increases above 1.2 , the respiration of the bacteroids decreases and the free O 2 concentration increases, which ultimately results in an inactivation of nitrogenase. From experiments with a H +-conducting ionophore, it is concluded that the lower rate of respiration at higher pH is caused by a higher intracellular ATP/ADP ratio. These observations demonstrate that the intracellular ATP/ADP ratio via the P i potential regulates the rate of respiration. This is similar with the classical mitochondrial respiratory control.In chapter 5 the ATP consumption by nitrogenase is compared with ATP synthesis by oxidative phosphorylation. The calculation shows that under conditions of nitrogen fixation the N 2 -reduction, is a major ATP-consuming process in the bacteroids. About 70 % of the ATP synthesized by oxidative phosphorylation is hydrolyzed by nitrogenase. Thus, nitrogenase by itself keeps the intracellular ATP/ADP ratio low and thereby stimulates the respiration.In chapter 6, the studied biochemical processes of the root nodule, are placed in a broader perspective. Four physiological processes in which malate is involved, are illuminated. A mechanism is postulated, which accounts for the balance between the supply of photosynthates and the supply of O 2 to the bacteroids. A change of the pH of the root nodule cells induced by changes of the malate concentration is the central theme of the proposal. The pH might influence the rate of respiration of the bacteroids and thus nitrogenase activity, but it also might regulate the O 2 influx into the central tissue of the root nodule. The pH changes are determined by the availability of sucrose for the root nodule cells. Finally the comparison between bacteroids and mitochondria is discussed.</TT
Practical applications of hydrogenase I from Pyrococcus furiosus for NADPH generation and regeneration
No full textThe soluble hydrogenase I (H-2:NADP(+) oxidoreductase, EC 1.18.99.1) from the marine hyperthermophilic strain of the archaeon Pyrococcus furiosus was partially purified by anion-exchange chromatography. This P furiosus hydrogenase I preparation (PF H(2)ase I) has been used as biocatalyst in the enzymatic production and regeneration of beta-1,4-nicotinamide adenindinucleotide phosphate, reduced form (NADPH), utilizing cheap molecular hydrogen and forming protons as the only side-product. Any excess of dihydrogen can be removed easily. It could be demonstrated, that this hyperthermophilic hydrogenase exhibits a high stability under reaction conditions. Generation as well as regeneration of NADPH were performed in batch and repetitive batch experiments with recyclisation of the biocatalyst. In two repetitive batch-series 6.2 g l(-1) NADPH could be produced with a total turnover number (ttn: mol produced NADPH/mol consumed enzyme) of 10,000. Utilizing the thermophilic NADPH-dependent alcohol dehydrogenase from Thermoanaerobium spec. (ADH M) coupled to the PF H(2)ase I in situ NADPH-regenerating system, two prochiral model substrates, acetophenone and (2S)-hydroxy-1-phenyl-propanone (HPP), were quantitatively reduced to the corresponding (S)-alcohol and (1R,2S)-diol. An e.e. >99.5% and d.e. >98%, respectively, with total turnover numbers (ttn: mol product/mol consumed cofactor NADP(+)) of 100 and 160 could be reached. (C) 2003 Elsevier B.V. All rights reserved
Practical applications of hydrogenase I from Pyrococcus furiosus for NADPH generation and regeneration
No full textThe soluble hydrogenase I (H-2:NADP(+) oxidoreductase, EC 1.18.99.1) from the marine hyperthermophilic strain of the archaeon Pyrococcus furiosus was partially purified by anion-exchange chromatography. This P furiosus hydrogenase I preparation (PF H(2)ase I) has been used as biocatalyst in the enzymatic production and regeneration of beta-1,4-nicotinamide adenindinucleotide phosphate, reduced form (NADPH), utilizing cheap molecular hydrogen and forming protons as the only side-product. Any excess of dihydrogen can be removed easily. It could be demonstrated, that this hyperthermophilic hydrogenase exhibits a high stability under reaction conditions. Generation as well as regeneration of NADPH were performed in batch and repetitive batch experiments with recyclisation of the biocatalyst. In two repetitive batch-series 6.2 g l(-1) NADPH could be produced with a total turnover number (ttn: mol produced NADPH/mol consumed enzyme) of 10,000. Utilizing the thermophilic NADPH-dependent alcohol dehydrogenase from Thermoanaerobium spec. (ADH M) coupled to the PF H(2)ase I in situ NADPH-regenerating system, two prochiral model substrates, acetophenone and (2S)-hydroxy-1-phenyl-propanone (HPP), were quantitatively reduced to the corresponding (S)-alcohol and (1R,2S)-diol. An e.e. >99.5% and d.e. >98%, respectively, with total turnover numbers (ttn: mol product/mol consumed cofactor NADP(+)) of 100 and 160 could be reached. (C) 2003 Elsevier B.V. All rights reserved
