scholarly journals The vanadium nitrogenase of Azotobacter chroococcum. Purification and properties of the VFe protein

1987 ◽  
Vol 244 (1) ◽  
pp. 197-207 ◽  
Author(s):  
R R Eady ◽  
R L Robson ◽  
T H Richardson ◽  
R W Miller ◽  
M Hawkins
Keyword(s):  
Nitrogenase Activity ◽  
Visible Region ◽  
Sedimentation Coefficient ◽  
Azotobacter Chroococcum ◽  
Acetylene Reduction Activity ◽  
Reduction Activity ◽  
Fe Protein ◽  
Purification And Properties ◽  
Mo Content ◽  
Ultracentrifugal Analysis

1. Nitrogenase activity of a strain of Azotobacter chroococcum lacking the structural genes for conventional nitrogenase (nifHDK) was separated into two components: an Fe-containing protein and a vanadoprotein. 2. The larger protein was purified to homogeneity by the criterion of electrophoresis of 10% (w/v) acrylamide gels in the presence of SDS. Two types of subunit, of Mr 50,000 and 55,000, were present in equal amounts. 3. The protein had an Mr of 210,000 and contained 2 V atoms, 23 Fe atoms and 20 acid-labile sulphide groups per molecule. The Mo content was less than 0.06 g-atom/mol. All the common amino acids were present, with a predominance of acidic residues. Ultracentrifugal analysis gave a maximum sedimentation coefficient of 9.7 S and a symmetrical boundary at 5 mg of protein X ml-1; dissociation occurred at lower concentrations. The specific activities (nmol of product/min per mg of protein), when assayed under optimum conditions with the complementary Fe protein from this strain, were 1348 for H2 evolution, 350 for NH3 formation and 608 for acetylene reduction. Activity was O2-labile, with a t1/2 of 40 s in air. At low temperatures the dithionite-reduced protein showed e.p.r. signals at g = 5.6, 4.35, 3.77 and 1.93, consistent with an S = 3/2 ground state with an additional S = 1/2 centre giving rise to the feature at g = 1.93. The u.v. spectra of dithionite-reduced and thionine-oxidized protein were very similar. Oxidation resulted in a general increase in absorbance in the visible region. The shoulder at 380 nm in the spectrum of reduced protein was replaced with shoulders near 330 nm and 420 nm on oxidation.

scholarly journals The vanadium nitrogenase of Azotobacter chroococcum. Purification and properties of the Fe protein

1988 ◽  
Vol 256 (1) ◽  
pp. 189-196 ◽  
Author(s):  
R R Eady ◽  
T H Richardson ◽  
R W Miller ◽  
M Hawkins ◽  
D J Lowe
Keyword(s):  
Nitrogenase Activity ◽  
Azotobacter Chroococcum ◽  
Blue Staining ◽  
Visible Range ◽  
Coomassie Blue Staining ◽  
Reduction Activity ◽  
Fe Protein ◽  
Coomassie Blue ◽  
Purification And Properties ◽  
Vanadium Nitrogenase

1. Nitrogenase activity of a strain of Azotobacter chroococcum lacking the structural genes of Monitrogenase (nifHDK) was associated with a V + Fe-containing protein and an Fe-containing protein [Robson, Eady, Richardson, Miller, Hawkins & Postgate (1986) Nature (London) 322, 388-390; Eady, Robson, Richardson, Miller & Hawkins (1987) Biochem. J. 244, 197-207]. 2. The Fe protein was purified to homogeneity by the criterion of Coomassie Blue staining after electrophoresis in 10% or 17% (w/v) polyacrylamide gels in the presence of SDS. One type of subunit, of Mr 32,000 +/- 2000, was found. 3. The native protein had an Mr of 62,500 +/- 2500 and contained approximately 4 Fe atoms and 4 acid-labile sulphide groups per molecule. The amino acid composition was similar to those of other purified Fe proteins, and, characteristically, tryptophan was absent. The specific activities (nmol of protein/min per mg of protein) when assayed under optimum conditions with the VFe protein from this strain were 1211 for H2 evolution under Ar, 337 for NH3 from N2 formation and 349 for C2H2 reduction. Activity of the Fe protein was O2-labile with a t1/2 of 36 s in air. At low temperatures the dithionite-reduced protein exhibited e.p.r. signals consistent with the presence of both S = 1/2 and S = 3/2 spin states. These signals were similar to those given by other nitrogenase Fe proteins, as were the changes in their line shape that occurred in the presence of MgATP or MgADP. The absorbance spectra showed that an increase in absorption occurred in the visible range on reversible oxidation of the dithionite-reduced protein. The oxidized-minus-reduced epsilon 420 was 6000 M-1.cm-1.

Nitrogenase Activity by Subterranean Clover and Other Temperate Pasture Legumes

1988 ◽  
Vol 15 (5) ◽  
pp. 657 ◽  
Author(s):  
AG Davey ◽  
RJ Simpson
Keyword(s):  
Root Respiration ◽  
Nitrogenase Activity ◽  
Subterranean Clover ◽  
Trifolium Subterraneum ◽  
Acetylene Reduction Activity ◽  
Diffusion Resistance ◽  
Pasture Legumes ◽  
Reduction Activity ◽  
Decline Rate ◽  
Nodulated Root

Nitrogenase (C2H2-reduction) activity and nodulated root respiration of intact plants of subterranean clover (Trifolium subterraneum L.) cv. Seaton Park nodulated by Rhizobium trifolii WU95 were measured in a flow-through system. Simultaneous declines in nitrogenase activity and respiration were exhibited 2 min after 10% C2H2 had been introduced into the gas stream. Declines in nitrogenase activity and nodulated root respiration provided an estimate of the efficiency of nitrogenase activity (mol CO2 evolved/mol C2H4 produced). The pre-decline rate of nitrogenase activity at time zero was thus calculated as the product of the respiration associated with nitrogenase activity and the reciprocal of the efficiency of nitrogenase activity. Pre-decline rates of nitrogenase activity were similar to peak rates for several pasture legumes. However, post-decline rates of activity were as much as 70% lower than the pre-decline rate. The age of subterranean clover plants had an important influence on the magnitude of the C2H2-induced decline; young plants exhibited the largest C2H2-induced inhibition of nitrogenase activity. Neither sainfoin (Onobrychis viciifolia Scop.) cv. Othello nodulated by Rhizobium sp. CC1108 nor yellow serradella (Ornithopus compressus L.) cv. Pitman nodulated by R. lupini WU425 exhibited C2H2-induced declines in nitrogenase activity. Nitrogenase-linked respiration of subterranean clover at the 14-leaf stage accounted for 50% of total nodulated root respiration. The oxygen diffusion resistance of the nodules increased in the presence of C2H2 but the effect was reversible once C2H2 was removed from the gas atmosphere. The pre-decline rate of acetylene reduction activity of subterranean clover reached a maximum at 10% C2H2. The C2H2-induced decline in nitrogenase activity was lower at subsaturating pC2H2 and was not detected at 0.4% C2H2.

A survey of angiosperms in Nova Scotia for rhizosphere nitrogenase (acetylene-reduction) activity

1978 ◽  
Vol 56 (18) ◽  
pp. 2218-2223 ◽  
Author(s):  
D. Smith ◽  
D. G. Patriquin
Keyword(s):  
Nova Scotia ◽  
Acetylene Reduction ◽  
Nitrogenase Activity ◽  
Reduction Technique ◽  
Acetylene Reduction Activity ◽  
Forage Grasses ◽  
Reduction Activity ◽  
Tropical Forage ◽  
Poorly Drained Soils

Excised root samples from 901 plants, representing 130 species of nonnodulated angiosperms largely in upland, pioneering habitats, were assayed for nitrogenase activity by the acetylene-reduction technique after overnight preincubation of the samples under low pO2. Most samples and most species exhibited very low excised root acetylene-reducing activities, but for 19 species, maximum values were greater than 50 nmol C2H4∙g−1∙h−1. In situ C2H2 assays, conducted on 10 species which had exhibited maximum excised root activities greater than 10 nmol C2H4∙g−1∙h−1, indicated average belowground N2-fixation rates of 3 to 92 g N∙ha−1∙day−1 and maxima greater than 100 g N∙ha−1∙day−1 for 3 of the 10 species. The highest values were for grasses characteristic of poorly drained soils and for some dicotyledonous weeds. It is concluded that the potential of temperate-zone angiosperms for nitrogenase activity by 'associative symbioses' approaches that of tropical forage grasses.

Temperature response of an associative N2-fixing Pseudomonas species in pure culture

1991 ◽  
Vol 37 (9) ◽  
pp. 715-718 ◽  
Author(s):  
Yiu-Kwok Chan
Keyword(s):  
Nitrogenase Activity ◽  
Temperature Response ◽  
Grass Species ◽  
Acetylene Reduction Activity ◽  
Optimum Growth Temperature ◽  
Reduction Activity ◽  
Key Words Nitrogen Fixation ◽  
Pseudomonas Species ◽  
Response To Temperature ◽  
Key Words Nitrogen

The growth rate and nitrogenase (acetylene reduction) activity of a pseudomonad isolated from the rhizosphere of the grass species Deschampsia caespitosa were investigated in the 0–35 °C temperature range. Growth was detected from 5 to 30 °C with or without provision of fixed N and its optimum growth temperature was about 25 °C, establishing its psychrotrophic response to temperature. Maximum nitrogenase activity was also found at about 25 °C. Low but significant nitrogenase activity was detected below 15 °C. This organism was confirmed to be a microaerobic diazotroph and it was sensitive to a dissolved O2 concentration of ca. 12 μM. It is probably dependent on the appropriate environment in the rhizosphere of the host plant in order to persist with sustained nitrogenase activity during cold and dormant periods. Key words: nitrogen fixation, Pseudomonas, psychrotroph, temperature.

scholarly journals Nitrogenase of Klebsiella pneumoniae: kinetics of formation of the transition-state complex and evidence for an altered conformation of MoFe protein lacking a FeMoco centre

1997 ◽  
Vol 326 (3) ◽  
pp. 637-640 ◽  
Author(s):  
Faridoon K. YOUSAFZAI ◽  
Robert R. EADY
Keyword(s):  
Klebsiella Pneumoniae ◽  
Transition State ◽  
Nitrogenase Activity ◽  
Active Species ◽  
Slow Phase ◽  
Mofe Protein ◽  
Fe Protein ◽  
Catalytically Active ◽  
Mo Content ◽  
Kinetics Of

We have investigated the kinetics of inactivation of Mo-nitrogenase isolated from Klebsiella pneumoniae when it forms an inhibited putative transition-state complex on incubation with ADP and AlF4-. In the presence of excess Kp2 (Fe protein of the Mo-nitrogenase of K. pneumoniae), the kinetics were found to depend on the Mo content of Kp1 (the MoFe protein of Mo-nitrogenase of K. pneumoniae). The residual nitrogenase activity versus time of incubation using Kp1 preparations containing integral, i.e. one or two Mo atoms per molecule of Kp1, were essentially monophasic, but significantly different rates of inactivation were observed. In contrast, the progress curves for preparations of Kp1 with non-integral Mo content were biphasic, suggesting the presence of two discrete catalytically active species of Kp1. The best fit to the observed data was obtained with a two-exponential expression, the amplitude of which was consistent with the Mo content, provided that the fast phase of the reaction was assigned to a Kp1 species containing one, and the slow phase to a species containing two Mo atoms per α2β2 tetramer. This analysis provides the first evidence for the existence of a catalytically active Kp1 species containing a single Mo atom. These data also indicate that MoFe protein which does not have all FeMoco binding sites occupied has an altered conformation compared with a fully loaded protein, and that the Fe protein reacts with these conformations at different rates to form the stable, but inhibited transition-state complex.

scholarly journals Molybdenum and vanadium nitrogenases of Azotobacter chroococcum. Low temperature favours N2 reduction by vanadium nitrogenase

1988 ◽  
Vol 256 (2) ◽  
pp. 429-432 ◽  
Author(s):  
R W Miller ◽  
R R Eady
Keyword(s):  
Low Temperature ◽  
Electron Flux ◽  
Specific Activity ◽  
Nitrogenase Activity ◽  
Azotobacter Chroococcum ◽  
Effect Of Temperature ◽  
High Electron ◽  
Mofe Protein ◽  
Fe Protein ◽  
Vanadium Nitrogenase

A comparison of the effect of temperature on the reduction of N2 by purified molybdenum nitrogenase and vanadium nitrogenase of Azotobacter chroococcum showed differences in behaviour. As the assay temperature was lowered from 30 degrees C to 5 degrees C N2 remained an effective substrate for V nitrogenase, but not Mo nitrogenase, since the specific activity for N2 reduction by Mo nitrogenase decreased 10-fold more than that of V nitrogenase. Activity cross-reactions between nitrogenase components showed the enhanced low-temperature activity to be associated with the Fe protein of V nitrogenase. The lower activity of homologous Mo nitrogenase components, although dependent on the ratio of MoFe protein to Fe protein, did not equal that of V nitrogenase even under conditions of high electron flux obtained at a 12-fold molar excess of Fe protein.

Relative contributions to nitrogenase (acetylene reducing) activity of stem and root nodules in Sesbania rostrata

1992 ◽  
Vol 38 (6) ◽  
pp. 577-583 ◽  
Author(s):  
J. K. Ladha ◽  
Minviluz Garcia ◽  
R. P. Pareek ◽  
G. Rarivoson
Keyword(s):  
Acetylene Reduction ◽  
Root Nodule ◽  
Nitrogenase Activity ◽  
Root Nodules ◽  
Dry Weight ◽  
Acetylene Reduction Activity ◽  
Sesbania Rostrata ◽  
Reduction Activity ◽  
Acetylene Reducing Activity ◽  
Aerial Stem

Six experiments, two each in the phytotron, greenhouse, and field, were conducted to assess the contribution of nitrogenase activity (acetylene reduction) by stem nodules in the presence and absence of root nodules of Sesbania rostrata (Brem & Oberm). In a greenhouse experiment, the effect of detaching already formed aerial stem nodules on the restoration of root nodules and nitrogenase activity was studied. The field experiment compared nodulation and acetylene-reduction activity by dual-nodulating S. rostrata and root-nodulating Sesbania cannabina. Acetylene-reduction activity expressed per gram of nodule dry weight was higher for stem nodules than for root nodules. Root nodule dry weight and acetylene-reduction activity failed to increase after stem inoculation, but root nodule dry weight and acetylene-reduction activity increased several fold within 15 days of detachment of aerial stem nodules. Stem nodulation, which occurred without inoculation under lowland field condition, suppressed root nodulation, thus accounting for more than 75% of total nitrogenase activity. Sesbania rostrata showed higher acetylene-reduction activity than S. cannabina. In dual-nodulating plants, root and stem nodules appeared to strike a balance in competition for energy, which may be controlled by stem nodulation. Key words: Sesbania rostrata, Azorhizobium caulinodans, stem nodule, root nodule, acetylene-reducing activity.

Stomatal conductance, photosynthesis and acetylene reduction rate in soybean genotypes

1992 ◽  
Vol 72 (2) ◽  
pp. 383-390 ◽  
Author(s):  
A. Djekoun ◽  
C. Planchon
Keyword(s):  
Glycine Max ◽  
Water Stress ◽  
Stomatal Conductance ◽  
Acetylene Reduction ◽  
Nitrogenase Activity ◽  
Leaf Water ◽  
Acetylene Reduction Activity ◽  
Reduction Activity ◽  
Leaf Osmotic Potential ◽  
Glycine Max L

Yield limitation in soybean (Glycine max L. Merr.) can result from decreases in photosynthesis and N2 fixation during periods of water deficiency. In this study, the relationships among stomatal conductance, photosynthesis and N2 fixation were analyzed in connection with drought tolerance of genotypes. Plants were grown in pots and exposed to field conditions. Carbon dioxide exchange rate was measured by gas analysis and nodule activity by the acetylene reduction method. Leaf water status was determined with a pressure bomb, and nodule water potential and leaf osmotic potential were measured psychrometrically. The differing tolerances of the cultivars Kingsoy and Hodgson to leaf water deficit resulted in a more or less developed ability of the lower side of the leaf to maintain good stomatal conductance during water stress. Stomatal conductance affects photosynthetic rate directly and acetylene reduction activity indirectly. Early stomatal closure, by limiting H2O exchange, contributes to conservation of nitrogenase activity. On the contrary, maintenance of high conductance during a water stress decreases soil water availability and nodule water content, which in turn has a decisive and limiting effect on acetylene reduction activity. Thus, if tolerance at low leaf water potentials associated with osmotic adjustment is an important drought mechanism for maintaining photosynthetic processes under water-limited conditions, the result would be obtained at the expense of symbiotic N2 fixation.Key words: Glycine max L. Merr., nitrogenase activity, photosynthesis, drought stress, soybean

Nitrogenase activity, nodulation and seed production inVicia fabaas affected by methabenzthiazuron

1996 ◽  
Vol 127 (3) ◽  
pp. 319-324 ◽  
Author(s):  
J. Martínez ◽  
D. Vidal ◽  
E. Simón
Keyword(s):  
Seed Production ◽  
Vegetative Growth ◽  
Nitrogenase Activity ◽  
Dry Weight ◽  
Inhibitory Effect ◽  
Acetylene Reduction Activity ◽  
N Recovery ◽  
N Fixation ◽  
N Content ◽  
Reduction Activity

SUMMARYThe effects of recommended application rates of the herbicide methabenzthiazuron (MBT) (2·5 and 4 kg ha-1) on vegetative growth, nodulation and N2fixation in faba bean plants were studied in the field at Barcelona during 1990 and 1991. Nodulation, specific nitrogenase activity (SNA) of excised nodules, dry weight of plants and seed nitrogen content at harvest were compared in MBT-treated, hand-weeded (HW) and control plants. In 1990 the higher MBT application (4 kg ha-1) caused a decrease in nodulation and acetylene reduction activity (ARA) per plant during the mid-period of pod-fill, and also in vegetative growth, seed production and N content of seeds, whereas the lower rate (2·5 kg ha-1) did not have a significant effect on any of these factors. However, in 1991, growth and herbicide effects were different from those in the previous year. No inhibitory effect of MBT on nodulation, SNA or ARA per plant was observed and vegetative growth, seed production and N content of seeds were not significantly different from those of plants from HW plots. In 1991, both 4 kg ha-1of MBT and the HW treatment caused a stimulatory effect on seed N recovery and yield compared to control plants. Rates of ARA per plant at late pod-fill were positively related to N recovery by seeds. These differences could have been due to the low rainfall in 1990, which may have permitted MBT to remain in the soil for a longer period and to inhibit N fixation and plant growth in theVicia fabacrop.

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