Role of Azotobacter vinelandii FdxN in FeMo-co biosynthesis

Biosynthesis of metal clusters for the nitrogenase component proteins NifH and NifDK involves electron donation events. Yet, electron donors specific to the biosynthetic pathways of the [4Fe–4S] cluster of NifH, or the P-cluster and the FeMo-co of NifDK, have not been identified. Here we show that an Azotobacter vinelandii mutant lacking fdxN was specifically impaired in FeMo-co biosynthesis. The ΔfdxN mutant produced 5-fold less NifB-co, an early FeMo-co biosynthetic intermediate, than wild type. As a consequence, it accumulated FeMo-co-deficient apo-NifDK and was impaired in NifDK activity. We conclude that FdxN plays a role in FeMo-co biosynthesis, presumably by donating electrons to support NifB-co synthesis by NifB. This is the first role in nitrogenase biosynthesis unequivocally assigned to any A. vinelandii ferredoxin.     

The relationship between substrate-induced respiration and swelling in Azotobacter vinelandii

1.

1. When oxidizable substrates are added to a starved suspension of Azotobacter vinelandii osmotically shrunken in 0.2 M KCl, a decrease in absorbance is observed which results from a change in light scattering as the cells increase in volume.     

Non-pyridine nucleotide dependent l-(+)-glutamate oxidoreductase in Azotobacter vinelandii

l-(+)-Glutamate oxidation that is non-pyridine nucleotide dependent is readily carried out by a membrane-bound enzyme in Azotobacter vinelandii strain O. Enzyme activity concentrates in a membranous fraction that is associated with the Azotobacter electron transport system. This l-glutamate oxidation is not dependent on externally added NAD⁺, NADP⁺, FAD, or FMN for activity. O₂, phenazine methosulfate and ferricyanide all served as relatively good electron acceptors for this reaction; while cytochrome c and nitrotetrazolium blue function poorly in this capacity. Paper chromatographic analyses revealed that the 2,4-dinitrophenylhydrazine derivative formed from the enzymatic oxidation of l-glutamate was α-ketoglutarate, while microdiffusion studies indicated that ammonia was also a key end product. These findings suggest that the overall reaction is an oxidative deamination. Ammonia formation was found to be stoichiometric with the amount of oxygen consumed (2 : 1 respectively, on a molar basis). The oxidation of glutamate was limited to the l-(+)-enantiomer indicating that this reaction is not the generalized type carried out by the l-amino acid oxidase. This oxidoreductase is functionally related to the Azotobacter electron transport system: (a) the activity concentrates almost exclusively in the electron transport fraction; (b) the l-glutamate oxidase activity is markedly sensitive to electron transport inhibitors, i.e. 2-n-heptyl-4-hydroxyquinoline-N-oxide, cyanide, and 4,4,4-trifluoro-1-(2-thienyl)-1,3-butanedione; and (c) spectral studies on the Azotobacter R₃ fraction revealed that a substantial amount of the flavoprotein (non-heme iron) and cytochrome (a₂, a₁, b₁, c₄ and c₅) are reduced by the addition of l-glutamate.     

Nitrogenase IV. Simple method of purification to homogeneity of nitrogenase components from Azotobacter vinelandii

Extracts of Azotobacter vinelandii have been fractionated by simple techniques to obtain highly purified components of nitrogenase. The yield of each component is greater than 60%. Purified Component I has a specific activity of 1638 nmoles ethylene formed/min per mg protein. The spectrum of Component I exhibits a broad absorption between 300 and 600 nm, with no distinctive peaks or shoulders. Addition of sodium dithionite or exposure to air has no effect on the absorption spectrum. Component I, examined at 4.2 °K has EPR signals at g = 4.2, 3.65 and 2.01. Addition of sodium dithionite does not produce additional resonances nor does it alter the signals already present. Crystals of Component I are dark brown and needle-shaped.Purified Component II has a specific activity of 1815 nmoles ethylene formed/min per mg protein. The absorption spectrum has no peaks or shoulders between 390 and 650 nm. Upon exposure of Component II to air, absorption increases between 400 and 650 nm. Treatment of oxidized Component II with dithionite causes this absorption to fall below that of the native Component II. EPR spectra of Component II has signals at g values of 2.05, 1.94, and 1.88. Upon inactivation by O₂, these signals disappear.Neither component by itself has detectable acetylene-reducing or N₂-fixing activity. The ratio of acetylene reduced to N₂ fixed is 3.86 with different ratios of the components. Both components form aggregated species upon exposure to air. Dithionite does not reverse this effect.     

Nitrogenase V The effect of Mo,W and V on the synthesis of nitrogenase components in Azotobacter vinelandii

When Azotobacter vinelandii OP is derepressed for nitrogenase synthesis in a medium containing no added Mo, component II but not component I is detected. Derepression with W in the medium allows the cells to produce inactive component I that can be activated by addition of molybdate to the medium. This activation does not require protein synthesis. Addition of V to Mo-free media does not increase the synthesis of component I protein. It is possible that Mo is an inducer of component I synthesis and that W also is capable of inducing component I, but that this component I is inactive because it lacks Mo.     

Oxidative phosphorylation in Azotobacter vinelandii. Energy-linked pH changes and fluorescence changes of atebrin and 1-anilinonaphthalene-8-sulphonate

1. Phosphorylating particles from Azotobacter vinelandii show a rapid, respiration-induced reversible increase in pH of the suspending medium; this is not found with non-phosphorylating particles.2. The observed pH response requires the presence of low concentrations of Mg²⁺ or of higher concentrations of Na⁺ or K⁺.3. Between 40 and 10 °C the rates of proton influx and efflux have similar temperature coefficients; below 10 °C the effect of temperature is greater on proton efflux.4. The kinetics of the energy-linked enhancement of fluorescence 1-anilinonaphthalene-8-sulphonate are slower than that of the quenching of the fluorescence of atebrin.     

The concentrations and thermodynamic activities of cations in intact Bryopsis chloroplasts

The internal cation levels of chloroplasts isolated from a green sea alga, Bryopsis maxima, were studied. Atomic absorption spectroscopy, combined with the determination of the sorbitol-impermeable and water-permeable spaces, revealed that chloroplasts contain an extremely high concentration of K⁺ and high levels of Na⁺, Mg²⁺ and Ca²⁺. A method was developed to estimate the thermodynamic activities of monovalent and divalent cations present in chloroplasts. pH changes induced by the addition of an ionophore (plus an H⁺ carrier), which makes the outer limiting membranes of chloroplasts permeable to both a cation and H⁺, were determined. Provided that the external pH was set equal to the internal pH, the internal concentration of the cation was estimated by determining the external cation concentration which gave rise to no electrochemical potential difference of the cation and hence no pH change on addition of the ionophore. The internal pH was determined by measuring distributions of radioactive methylamine and 5,5-dimethyloxazolidine-2,4-dione between the chloroplast and medium (Heldt, H.W., Werdan, K., Milovancev, M. and Geller, G. (1973) Biochim. Biophys. Acta 314, 224–241). The internal pH was also estimated by measuring pH changes caused by the disruption of the outer limiting membrane with Triton X-100. The results indicate that a significant part of the monovalent cations and most of the divalent cations are attracted into a diffuse layer adjacent to the negatively charged surfaces of membranes and proteins, or form complexes with organic and inorganic compounds present in the intact chloroplasts.     

Antioxidant metabolism of grapefruit infected with Xanthomonas axonopodis pv. citri

Grapefruit is one of the most susceptible citrus genotypes to Asiatic Citrus Canker, caused by Xanthomonas axonopodis pv. citri (Xac), that can cause severe losses in citrus yield and quality. Although much is known about citrus response to Xac, little is known of the role of antioxidant metabolism. Grapefruit leaves were artificially injected with a strain of Xac obtained from a commercial grove in Florida and components of oxidative metabolism were measured. Symptoms observed included water soaking (2 dai; days after inoculation), raised and ruptured epidermis (6-8 dai), formation of necrotic lesions (16 dai), and leaf abscission (21 dai). The Xac population increased to a maximum (≈10⁹ CFU/cm²) 8 dai and then declined to ≈10⁷ CFU/cm² by 20 dai. Lipid peroxidation was higher in infected leaves than uninoculated controls from 4 to 21 dai indicating greater oxidative stress. H₂O₂ concentration demonstrated a biphasic pattern with peak concentrations at 4 and 13 dai and minimum concentrations that were lower than the controls at 10 and 20 dai. The H₂O₂ concentration somewhat corresponded with superoxide dismutase (SOD) activity, which generates H₂O₂ via dismutase of superoxide ions. Total SOD activity in Xac-infected leaves increased to a maximum at 4 dai, the day of highest H₂O₂ concentration, and then declined and remained at or below controls. Mn-SOD and Fe-SOD activities both increased to maximum activities at 4 dai. Mn-SOD had four isoforms in Xac-infected leaves but only three in the controls. Fe-SOD had three isoforms in both infected and control plants. Suppression of H₂O₂ in Xac-infected leaves also corresponded to higher activities of the H₂O₂ catabolising enzymes catalase (CAT), ascorbate peroxidase (APOD), and peroxidase (POD). Two additional CAT isoforms were detected in infected leaves and not the controls. Three POD isoforms were detected in both control and infected leaves. Previous research has shown that Xac is sensitive to intraplant H₂O₂ concentration, however, the pattern of Xac in this study did not correspond to H₂O₂ concentration, which initially increased due to enhanced SOD activity, but was later suppressed apparently with the aid of peroxidases. In conclusion, Xac infection altered H₂O₂ metabolism in grapefruit leaves by changes in the activities and isoforms of SODs, CATs, PODs and APOD.     

Purine metabolism in Leishmania donovani and Leishmania braziliensis

We have studied purine metabolism in the culture forms of Leishmania donovani and Leishmania braziliensis. These organisms are incapable of synthesizing purines de novo from glycine, serine, or formate and require an exogenous purine for growth. This requirement is better satisfied by adenosine or hypoxanthine than by guanosine. Bothe adenine and inosine are converted to a common intermediate, hypoxanthine, before transformation to nucleotides. This is due to the activity of an adenine aminohydrolase (EC 3.5.4.2), a rather unusual finding in a eukaryotic cell. There is a preferential synthesis of adenine nucleotides, even when guanine or xanthine are used as precursors.The pathways of purine nucleotide interconversions in these Leishmania resemble those found in mammalian cells except for the absence of de novo purine biosynthesis and the presence of an adenine-deaminating activity.     

Regulation of dinitrogen fixation in intact Azotobacter vinelandii

1. In intact Azotobacter vinelandii the influence of oxygen on the levels of oxidized nicotinamide adenine dinucleotides and adenine nucleotides in relation to nitrogenase activity was investigated.

2. The hypothesis that a high (NADH + NADPH)/(NAD⁺ + NADP⁺) is the driving force for the transport of reducing equivalents to nitrogenase in intact A. vinelandii was found to be invalid. On the contrary, with a decreasing ratio of reduced to oxidized pyridine nucleotides, the nitrogenase activity of the whole cells increases.

3. By measuring oxidative phosphorylation and using 9-amino acridine as a fluorescent probe, it could be demonstrated that respiration-coupled transport of reducing equivalents to the nitrogenase requires a high energy level of the plasma membrane or possibly coupled to it, a high pH gradient over the cytoplasmic membrane. Furthermore nitrogen fixation is controlled by the presence of oxygen and the ATP/ADP ratio.     

Phytate metabolism in Petunia pollen

Phytic acid has been detected in the anthers of young flower buds of Petunia hybrida, the amount increasing slowly as the flower develops until anther dehydration, when there was a more rapid increase in phytic acid content. In mature pollen, the phytic acid content was found to be 2.0 % by weight, of which 90 % was water soluble, while free myo-inositol was a relatively low 0.06 % by weight. Breakdown of phytic acid was initiated soon after pollen germination began, and its degradation products, myo-inositol and inorganic phosphate, were rapidly mobilized for phospholipid and pectin biosynthesis. Both are in high demand during pollen tube elongation. Utilization of myo-[2-³H]inositol for phospholipid biosynthesis was about five times that for pectin synthesis during the first few hours of pollen germination. The label in the phospholipid was identified as the myo-inositol moiety of phosphaltidylinositol, while the pectin material contained predominantly labelled arabinose, with smaller amounts of label in galacturonic acid, glucose and xylose. A chase experiment showed that the myo-inositol moiety of phosphatidylinositol was subject to a relatively rapid turnover, while the label in pectin was not. Labelling germinating pollen with [³²P]orthophosphate gave label in phosphatidic acid, phosphatidylinositol, phosphatidylethanolamine and phosphatidylcholine of the phospholipids. Phosphatidylinositol contained 30 % of this label initially, a proportion which declined to 10 % over longer periods of germination.