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Reduced Nicotinamide-Adenine Dinucleotide-Nitrite Reductase from Azotobacter Chroococcum by J

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Reduced Nicotinamide-Adenine Dinucleotide-Nitrite Reductase from Azotobacter Chroococcum by J Biochem. J. (1973) 133, 701-708 701 Printed in Great Britain Reduced Nicotinamide-Adenine Dinucleotide-Nitrite Reductase from Azotobacter chroococcum By J. M. VEGA, M. G. GUERRERO, E. LEADBETTER* and M. LOSADA Departamento de Bioquimica, Facultad de Ciencias y Consejo Superior de Investigaciones Cientificas, Universidad de Sevilla, Seville, Spain (Received 12 February 1973) 1. The assimilatory nitrite reductase of the N2-fixing bacterium Azotobacter chroococcum was prepared in a soluble form from cells grown aerobically with nitrate as the nitrogen source, and some of its properties have been studied. 2. The enzyme is a FAD-dependent metalloprotein (mol.wt. about 67000), which stoicheiometrically catalyses the direct reduction ofnitrite to NH3 with NADH as the electron donor. 3. NADH-nitrite reductase can exist in two either active or inactive interconvertible forms. Inactivation in vitro can be achieved by preincubation with NADH. Nitrite can specifically protect theenzyme against this inactivation and reverse the process once it has occurred. 4. A. chroococcum nitrite reductase is an adaptive enzyme whose formation depends on the presence of either nitrate or nitrite in the nutrient solution. 5. Tungstate inhibits growth of the micro- organism very efficiently, by competition with molybdate, when nitrate is the nitrogen source, but does not interfere when nitrite or NH3 is substituted for nitrate. The addition of tungstate to the culture media results in the loss of nitrate reductase activity but does not affect nitrite reductase. The enzyme involved in the assimilatory reduction reduction of nitrite by the extracts was identified as of nitrite to NH3 in algae and higher plants has been NH3, whereas that of hydroxylamine reduction was thoroughly characterized in recent years and classi- not established. Both nitrite reductase and hydroxyl- fied as ferredoxin-nitrite reductase (Beevers & Hage- amine reductase were adaptive enzymes, their forma- man, 1969; Hewitt, 1970; Kessler, 1971; Losada, tion being stimulated by nitrate. Escherichia coli 1972). Nitrite reductase purified to homogeneity strain Bn grown in deep standing cultures with nitrate from green cells of different sources (Chlorella, as the sole source of nitrogen has been shown to con- spinach and squash leaves) contains 2 atoms of non- tain at least three nitrite reductases that reduced haem iron/molecule of 63000 daltons and does not nitrite (and hydroxylamine) to NH3 (Lazzarini & seem to be a flavoprotein (Losada & Paneque, 1971; Atkinson, 1961), but only the enzyme specific for Cardenas et al., 1972a,b; Zumft, 1972). On the other NADH appears to be responsible for physiological hand, the enzyme isolated from the nitrate-assimilat- nitrite reduction (Kemp & Atkinson, 1966). Zarowny ing fungi Neurospora crassa (Nason et al., 1954; & Sanwal (1963) have also observed a NADH- Nicholas et al., 1960) and Torulopsis nitratophila specific nitrite reductase in extracts of E. coli K-12 (Rivas et al., 1973) has been characterized as an grown with nitrate as nitrogen source. Prakash & NAD(P)H-nitrite reductase which specifically re- Sadana (1972) have obtained from Achromobacter quires FAD and some metal component(s). fisheri grown on nitrate under low 02 partial pressure By contrast with nitrite reductases from the plant a homogenous haemoprotein that catalyses the reduc- kingdom, information on nitrite reductase from bac- tion of nitrite (and hydroxylamine) to NH3 and have teria of the assimilatory nitrate-reducing type is very pointed out that any biosynthetic function of the scanty (Nason, 1962; Takahashi et al., 1963; Hewitt enzyme appears fortuitous. & Nicholas, 1964). Spencer et al. (1957) found in ex- The present paper describes the preparation and tracts of Azotobacter vinelandii a soluble nitrite and characterization ofa soluble NADH-nitrite reductase hydroxylamine reductase system, which utilized re- of the assimilatory type from Azotobacter chroococ- duced nicotinamide nucleotides as electron donors cum cells aerobically grown on nitrate as the nitrogen and required added flavin nucleotides for maximal source. The enzyme is a FAD-dependent metallo- activity. Inhibitor studies indicated that the system protein(mol.wt. about 67000) of adaptive nature that had an essential metal component. The product ofthe stoicheiometrically catalyses the direct reduction of * Present address: Department of Biology, Amherst nitrite to NH3 and exists in either active or inactive College, Amherst, Mass. 01002, U.S.A. interconvertible forms. Vol. 133 702 J. M. VEGA, M. G. GUERRERO, E. LEADBETTER AND M. LOSADA Materials and Methods 2,umol; an appropriate amount of enzyme pre- Cell culture paration. Although high-speed centrifugation prac- tically removed the NADH oxidase activity originally A. chroococcum (strain A.T.C.C. 4412, from Sala- present in the crude extracts, NADH-nitrite reduc- manca University's culture collection) was grown tase activity was always corrected for the rate of under air with vigorous shaking at 27°C on a syn- NADH oxidation in the absence of nitrite. Reduced thetic medium containing (mmol/litre): mannitol, 39; Methyl Viologen-nitrite reductase was determined by KNO3, 8; MgSO4,7H20, 0.8; CaCl2, 2H20, 0.34; nitrite disappearance (Ramirez et al., 1966), but with FeSO4,7H20, 0.19; NaCl, 3.4; Na2MoO4, 0.01; potassium phosphate buffer, pH7.0, instead of Tris- disodium EDTA, 0.02; potassium phosphate buffer, HC1 buffer. One unit of activity corresponds to the pH7.3, 10. When NH3 or NH4NO3 replaced KNO3 oxidation of 1 ,tmol ofNADH (or the stoicheiometric as the source ofnitrogen, total concentration ofnitro- reduction of ijmol of nitrite)/min. gen was also maintained at 8mM. Since nitrite was Nitrate reductase activity was measured by colori- toxic at concentrations higher than 1 mm, care was metrically following the appearance of nitrite. The taken not to exceed this limit when NaNO2 or reaction mixture was incubated at 30°C for 2min and NaNO2 plus (NH4)2S04 was the nitrogen source. In contained, in a final volume of 1 ml: potassium phos- experiments involving the effect of tungstate, molyb- phate buffer, pH7.0, 100,umol; KNO3, 10,umol; date was omitted from the standard nutrient solution Methyl Viologen, 0.15,umol; Na2S204 in 0.1ml of and tungstate was added as indicated; the cells used 95mM-NaHCO3, 0.8mg; an appropriate amount for inoculation in these experiments were grown on of enzyme preparation. The reaction was stopped by nitrate media lacking added molybdate. vigorouslyshaking the assaymixture. Enzyme activity units are expressed as jumol of nitrite formed/min. Cell-free extracts Cells (lOg fresh wt.) were harvested in the exponen- Analytical methods tial phase of growth by low-speed centrifugation, Growth was determined by measuring absorbance washed with 50mM-potassium phosphate buffer, changes at 660nm. Nitrite was measured by the pH 7.0, and resuspended in 3ml of the same buffer. method of Snell & Snell (1949), and nitrate with The crude extract was prepared by breaking the cells nitrate reductase from spinach leaves as described by at 2°C for 2min in a vibration homogenizer (Buhler) Relimpio et al. (1972). NH3 was determined by treat- with 100ml of glass beads (0.1mm diam.). The bro- ment with Nessler's reagent after diffusion and ken material was extracted with 50ml of the absorption of the gas in 5mM-H2SO4 in Conway phosphate buffer and after centrifugation for 20min units (Conway, 1957). Protein was measured by the at 40000g the supernatant was used as enzyme source method of Lowry et al. (1951) with ovalbumin as a for the preparation of nitrite reductase. standard. The molecular weight of NADH-nitrite reductase Nitrite reductase was estimated by sucrose-density-gradient centri- Since the crude extract possessed high NADH oxi- fugation (Martin & Ames, 1961). Samples (0.2ml) of dase activity, it was centrifuged at 2°C in a L2-50B nitrite reductase and several other proteins of known Spinco ultracentrifuge for 3h at 120000g and the molecular weight (catalase, 240000; glucose 6-phos- resulting supernatant, which was practically free of phate dehydrogenase, 110000; serum albumin, oxidase activity, was used as the nitrite reductase 67000) were layered on the top of a 3.4ml linear preparation. The enzyme was unusually unstable 5-20% (w/v) sucrose gradient layered over 0.1 5ml of when purification was attempted. 50% (w/v) sucrose. Sucrose was dissolved in 50mM- potassium phosphate buffer, pH 7.0. Centrifugation Enzyme assays was carried out at 45000 rev./min for 11 h at 2°C in a Spinco L2-50B ultracentrifuge with a SW-56 Ti rotor. Two different methods were used for the assay of Gradients were fractioned from the bottom of the nitrite reductase activity. NADH-nitrite reductase tube with a Densi-Flow apparatus from Buchler was measured by following the nitrite-dependent oxi- Instruments Inc., Fort Lee, N.J., U.S.A. Fractions dation ofNADH. The reaction was carried out under (3 drops) were collected with a LKB fraction col- air at room temperature and absorbance at 340nm lector. Protein and enzyme activities were then deter- was recorded with a Beckman DK-2A spectrophoto- mined in meter, or exceptionally (for the measurement of the samples of each fraction. K. value for NADH) fluorescence at 450nm was re- corded with an Aminco-Bowman 4-8202 spectro- Results photofluorimeter. The reaction mixture included, in a Electron donors and cofactors final volume of 2ml: potassium phosphate buffer, Table 1 shows that NADH was an effective electron pH7.0. 150,mol; NADH, 0.3,umol; NaNO2, donor for the reduction of nitrite catalysed by A. 1973 NADH-NITRITE REDUCTASE FROM AZOTOBACTER CHROOCOCCUM 703 Table 1. Electron donors and cofactors for nitrite chroococcum nitrite reductase. The reaction was reductasefrom A. chroococcum specifically stimulated about twofold by the addition In the of FAD; FMN was without effect. NADPH was a experiments with reduced nicotinamide nucleo- relatively ineffective electron donor compared with tides as electron donors, the reaction mixture con- NADH tained, inafinal volume of2ml: potassium phosphate and the addition of FAD gave no additional buffer, stimulation.
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