scholarly journals Crystal structure of the [2Fe–2S] protein I (Shethna protein I) from Azotobacter vinelandii

2021 ◽  
Vol 77 (11) ◽  
Author(s):  
Burak V. Kabasakal ◽  
Charles A. R. Cotton ◽  
James W. Murray
Keyword(s):  
Crystal Structure ◽  
Nitrogen Fixation ◽  
Azotobacter Vinelandii ◽  
Nitrogenase Activity ◽  
Redox Chemistry ◽  
Evolutionary Relationships ◽  
Cysteine Residues ◽  
Aquifex Aeolicus ◽  
Iron Sulfur ◽  
The 1960S

Azotobacter vinelandii is a model diazotroph and is the source of most nitrogenase material for structural and biochemical work. Azotobacter can grow in above-atmospheric levels of oxygen, despite the sensitivity of nitrogenase activity to oxygen. Azotobacter has many iron–sulfur proteins in its genome, which were identified as far back as the 1960s and probably play roles in the complex redox chemistry that Azotobacter must maintain when fixing nitrogen. Here, the 2.1 Å resolution crystal structure of the [2Fe–2S] protein I (Shethna protein I) from A. vinelandii is presented, revealing a homodimer with the [2Fe–2S] cluster coordinated by the surrounding conserved cysteine residues. It is similar to the structure of the thioredoxin-like [2Fe–2S] protein from Aquifex aeolicus, including the positions of the [2Fe–2S] clusters and conserved cysteine residues. The structure of Shethna protein I will provide information for understanding its function in relation to nitrogen fixation and its evolutionary relationships to other ferredoxins.

Nitrogen fixation

1976 ◽  
Vol 274 (934) ◽  
pp. 341-358 ◽  
Keyword(s):  
Nitrogen Fixation ◽  
Metabolic Regulation ◽  
Higher Plants ◽  
Focal Point ◽  
Nitrogenase Activity ◽  
Nitrogen Fixing ◽  
Blue Green Algae ◽  
International Biological Programme ◽  
The 1960S ◽  
Biological Programme

The International Biological Programme served as a focal point for studies on biological nitrogen fixation during the 1960s. The introduction of the acetylene reduction technique for measuring nitrogenase activity in the field led to estimates becoming available of the contribution of lichens, blue-green algae, nodulated non-legumes and bacterial-grass associations, as well as of legumes. Other studies carried out on the physiology and biochemistry of the process led to the eventual purification and characterization of the nitrogenase enzyme. These studies, collectively, provided the springboard for current work, so essential in view of the present energy crisis, on how to increase the use and efficiency of nitrogen-fixing plants, on the metabolic regulation of the nitrogenase enzyme and on the genetics of the nitrogen-fixing process, both in higher plants and in free-living micro-organisms.

para-Hydroxybenzoate supported nitrogen fixation in Azotobacter vinelandii strain OP (13705)

1988 ◽  
Vol 34 (11) ◽  
pp. 1271-1275 ◽  
Author(s):  
Jay B. Peterson ◽  
Lynn S. Peterson
Keyword(s):  
Nitrogen Fixation ◽  
Cytoplasmic Membrane ◽  
Azotobacter Vinelandii ◽  
Nitrogenase Activity ◽  
Molecular Nitrogen ◽  
Carbon Sources ◽  
Gel Filtration ◽  
Cell Extracts ◽  
Reduction Assay ◽  
Membrane Bound

Azotobacter vinelandii cells grew with molecular nitrogen and p-hydroxybenzoate as the sole added nitrogen and carbon sources. Nitrogenase activity in p-hydroxybenzoate grown cells was demonstrated with the acetylene reduction assay. Cell extracts contained the enzymes p-hydroxybenzoate hydroxylase (EC 1.14.13.2) and protocatechuate 3,4-dioxygenase (EC 1.13.1.3); oxygenases associated with p-hydroxybenzoate metabolism. These enzymes separated from respiration particles with gel filtration chromatography, indicating that they are soluble and not membrane bound. This evidence indicates that oxygen enters to the inner face of the cytoplasmic membrane during nitrogen fixation.

Construction of recombinant Escherichia coli producing nitrogenase-related proteins from Azotobacter vinelandii

2021 ◽  
Author(s):  
Yuki Tatemichi ◽  
Takeharu Nakahara ◽  
Mitsuyoshi Ueda ◽  
Kouichi Kuroda
Keyword(s):  
Escherichia Coli ◽  
Nitrogen Fixation ◽  
Biological Nitrogen Fixation ◽  
Fossil Fuels ◽  
Azotobacter Vinelandii ◽  
Nitrogenase Activity ◽  
Gene Clusters ◽  
Fixation Method ◽  
Overlap Extension ◽  
Biological Nitrogen

Abstract Biological nitrogen fixation by nitrogenase has attracted attention as an alternative method to chemical nitrogen fixation, which requires large amounts of fossil fuels. Azotobacter vinelandii, which produces an oxygen-sensitive nitrogenase, can fix nitrogen even under aerobic conditions; therefore, the heterologous expression of nif-related genes from A. vinelandii is a promising strategy for developing a biological nitrogen fixation method. We assembled 17 nif-related genes, which are scattered throughout the genome of A. vinelandii, into synthetic gene clusters by overlap-extension-PCR and seamless cloning and expressed them in Escherichia coli. The transcription and translation of the 17 nif-related genes were evaluated by RT-qPCR and LC-MS/MS, respectively. The constructed E. coli showed nitrogenase activity under anaerobic and microaerobic conditions. This strain would be a useful model for examining the effect of other genes from A. vinelandii on nitrogen fixation by expressing them in addition to the minimal set of nif-related genes.

Molybdenum enhancement of nitrogen fixation in a Mo-starved Azotobacter vinelandii Nif− mutant

1982 ◽  
Vol 28 (10) ◽  
pp. 1173-1180 ◽  
Author(s):  
William J. Page ◽  
S. Karen Collinson
Keyword(s):  
Molecular Weight ◽  
Nitrogen Fixation ◽  
Azotobacter Vinelandii ◽  
Nitrogenase Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Nitrogen Fixing ◽  
Wild Type ◽  
Cell Extracts ◽  
In The Wild ◽  
Dinitrogenase Reductase

Molybdenum (Mo)-starved wild-type and Nif− strains of Azotobacter vinelandii reduced acetylene (fixed nitrogen) in Mo-limited nitrogen-free medium. Vanadate enhanced this activity in all of the strains. Molybdate caused repression of nitrogenase activity in the Nif− mutants and enhanced the nitrogenase activity in the wild type. The nitrogenase activity in the Nif− mutant UW3, however, was enhanced by Mo, became maximal after 3 h, and then declined to zero after 10 h of incubation. The activation of nitrogenase by Mo followed a 5- to 10-min lag and was inhibited when streptomycin or rifampin was added with Mo. Examination of Mo-starved nitrogen-fixing UW3 cell extracts by two-dimensional polyacrylamide gel electrophoresis revealed molecular weight 57 000, 50 000, and 30 000 proteins that were Mo and NH4+ repressive. The molecular weight 30 000 protein appeared in the same position on the gel as the wild-type dinitrogenase reductase, although UW3 did not produce this protein under Mo-sufficient nitrogen-fixing conditions. Cell extracts prepared 3 h after Mo addition lacked the molecular weight 57 000 and 50 000 proteins but contained a new protein corresponding to the β subunit of dinitrogenase. When UW3 nitrogenase activity was lost, the dinitrogenase reductase-like protein also was absent. The results suggest that a complex active in nitrogen fixation may form between components of the traditional Mo-sufficient and alternative Mo-starved cell nitrogen fixation systems.

Respiratory Protection of Nitrogenase Activity in Azotobacter vinelandii—Roles of the Terminal Oxidases

1997 ◽  
Vol 17 (3) ◽  
pp. 303-317 ◽  
Author(s):  
Robert K. Poole ◽  
Susan Hill
Keyword(s):  
Gene Expression ◽  
Nitrogen Fixation ◽  
Oxygen Reduction ◽  
Crucial Role ◽  
Azotobacter Vinelandii ◽  
Nitrogenase Activity ◽  
Terminal Oxidase ◽  
Respiratory Protection ◽  
Terminal Oxidases ◽  
Cytochrome Bd

Nitrogen fixation by aerobic prokaryotes appears paradoxical: the nitrogen-fixing enzymes—nitrogenases—are notoriously oxygen-labile, yet many bacteria fix nitrogen aerobically. This review summarises the evidence that cytochrome bd, a terminal oxidase unrelated to the mitochondrial and many other bacterial oxidases, plays a crucial role in aerotolerant nitrogen fixation in Azotobacter vinelandii and other bacteria by rapidly consuming oxygen during uncoupled respiration. We review the pertinent properties of this oxidase, particularly its complement of redox centres, the catalytic cycle of oxygen reduction, the affinity of the oxidase for oxygen, and the regulation of cytochrome bd gene expression. The roles of other oxidases and other mechanisms for limiting damage to nitrogenase are assessed.

Studies on compatibility of nitrogen fixation by high-temperature-adapted Rhizobium strains and Vigna radiata genotypes at two moisture levels in calcareous soil

1983 ◽  
Vol 101 (2) ◽  
pp. 377-381 ◽  
Author(s):  
R. Rai ◽  
V. Prasad
Keyword(s):  
Nitrogen Fixation ◽  
High Temperature ◽  
Grain Yield ◽  
Vigna Radiata ◽  
Calcareous Soil ◽  
Nitrogenase Activity ◽  
Summer Season ◽  
Green Gram ◽  
Rhizobium Strains

SUMMARYRhizobium strains adapted to high temperature, and genotypes of green gram, were used to study the symbiotic N2-fixation in a summer season at two moisture levels in calcareous soil. Different interactions between strains and genotypes were observedatthe two moisture levels. At both moisture levels, strain S4 with the green gram genotype S8 showed the greatest grain yield, nitrogenase activity, leghaemoglobin and ethanolsoluble carbohydrate of nodules.

Nitrogen fixation by cell-free extracts of Klebsiella pneumoniae

1968 ◽  
Vol 14 (1) ◽  
pp. 33-38 ◽  
Author(s):  
M. C. Mahl ◽  
P. W. Wilson
Keyword(s):  
Nitrogen Fixation ◽  
Klebsiella Pneumoniae ◽  
Azotobacter Vinelandii ◽  
Ammonium Ion ◽  
Michaelis Constant ◽  
Nitrogen Gas ◽  
Aerobacter Aerogenes ◽  
Free System ◽  
Evolving System ◽  
A Cell

A cell-free system which permits nitrogen fixation by extracts of Klebsiella pneumoniae M5al (formerly Aerobacter aerogenes) has been developed. It is, essentially, that system described by Bulen and associates for Azotobacter vinelandii, utilizing ATP as a source of energy and dithionite as a source of electrons. The Michaelis constant for fixation has been estimated to be 0.12 atm. The extracts possessed an ATP-dependent hydrogen evolving system. Hydrogen evolution from these extracts was less under nitrogen than under helium in the presence of ATP. Nitrogen gas appears to be the inducer of nitrogen fixation. In the absence of N2, no induction of nitrogenase occurs. Nitrogenase is absent in cells grown on NH4+-N. There is a lag of about 13 h after the introduction of N2 gas into a culture which has depleted its supply of NH4+-N before nitrogenase can be detected. For reasons discussed in the text, this conclusion must be regarded as tentative at this time. Ammonium ion appears to prevent the synthesis of new molecules of nitrogenase without affecting the activity of those already formed.

Formation of the nitrogen-fixing enzyme system in Azotobacter vinelandii

1968 ◽  
Vol 14 (1) ◽  
pp. 25-31 ◽  
Author(s):  
G. W. Strandberg ◽  
P. W. Wilson
Keyword(s):  
Color Change ◽  
Azotobacter Vinelandii ◽  
Nitrogenase Activity ◽  
Color Difference ◽  
Potassium Nitrate ◽  
Whole Cells ◽  
System A ◽  
Growth System ◽  
A Cell ◽  
Enzyme Formation

The formation and activity of nitrogenase2 in Azotobacter vinelandii OP was examined using a cell-free assay system. A lag period of about 30 min occurred between the exhaustion of the combined nitrogen source and growth on N2. Cells grown on ammonium acetate or potassium nitrate had no detectable nitrogenase activity. Nitrogenase activity appeared in cells, grown under a flowing gas phase of 20% O2 – 60% He, about 45 min after the exhaustion of ammonia. Nitrogenase formation was inhibited in a closed system with an atmosphere containing 40% O2 but not by one containing 20% O2. Hydrogen did not inhibit enzyme formation. The question of whether N2 is required for the formation of the enzyme could not be answered as this gas could not be completely eliminated from the growth system. Chloramphenicol prevented the formation of the enzyme and inhibited nitrogen fixation in whole cells, but had no effect on cell-free enzyme activity. A brief rise in turbidity which occurred during nitrogenase formation appeared to be due to a color change in the cells from reddish brown to dark brown. Spectrophotometric examination of extracts from ammonia- and N2-grown cells did not reveal any components responsible for this color difference, but this result may reflect only the presence of interfering substances in the crude extract.

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