Formation of the nitrogen-fixing enzyme system in <i>Azotobacter vinelandii</i>

Strandberg, G.W.; Wilson, Paul W.

doi:10.1139/m68-005

Cited by 293 publications

(137 citation statements)

References 5 publications

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“…Both our work and that of Kelly (1969a, b) are consistent with the report of Strandberg & Wilson (1968) that prolonged (4 h.) shaking of A . vinelandii cultures in air decreased N,-fixing activity of extracts 28-fold and that synthesis of nitrogenase by NH,+-limited A .…”

Section: Reversible Oxygen Inhibition and 'Conformational Protection 'supporting

confidence: 93%

“…Our experiments with chloramphenicol, which inhibits nitrogenase synthesis in Azotobacter vinvlandii (Strandberg & Wilson, 1968) support this view. Rapidly reversible 0, inhibition of nitrate reductase activity, also without protein synthesis, occurs in anaerobically grown cultures of Aerobacter aerogenes (Pinchinoty,I 963) and the tetrathionate reductase activity of a similarly grown unidentified coliform bacterium (Pinchinoty & Bigliardi-Rouvier, 1963) is also oxygen-sensitive.…”

Section: Reversible Oxygen Inhibition and 'Conformational Protection 'supporting

confidence: 65%

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Effects of Oxygen on Acetylene Reduction, Cytochrome Content and Respiratory Activity of Azotobacter chroococcum

Drozd

Postgate

1970

Journal of General Microbiology

131

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S U M M A R YThe respiratory activities and cytochrome a2 contents of nitrogen-fixing continuous cultures of Azotobacter chroococcum (NCIB 8003) increased with the partial pressure of oxygen encountered during growth. Above 0.6 atm., wash-out of the culture occurred. Acetylene reduction by culture samples of low respiratory activity was far more easily inhibited by oxygenation than was that of samples of high respiratory activity, though their maximum acetylenereducing activities at their optimal pOz values were similar. Inhibition by oxygen was reversible after mild oxygenation: 70 to IOO % of the original activity returned immediately when the degree of oxygenation was decreased. Irreversible inhibition occurred after vigorous oxygenation and was associated with a loss of activity in cell-free extracts, which was restored by adding the oxygen-sensitive protein component of Azotobacter nitrogenase. These observations support earlier proposals that augmented respiration can scavenge oxygen from the nitrogen-fixing site and that a conformational change in the state of nitrogenase can prevent damage to the enzyme by oxygen. Vigorous aeration, however, may overcome these protective mechanisms.

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Section: Reversible Oxygen Inhibition and 'Conformational Protection 'supporting

confidence: 93%

Section: Reversible Oxygen Inhibition and 'Conformational Protection 'supporting

confidence: 65%

Effects of Oxygen on Acetylene Reduction, Cytochrome Content and Respiratory Activity of Azotobacter chroococcum

Drozd

Postgate

1970

Journal of General Microbiology

131

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“…Isolation of Acetylene-resistant Strains and DNA BiochemistryStrains of A. vinelandii used in this study were grown under diazotrophic (nitrogen-fixing) conditions using a modified Burk medium previously described (18). Spontaneous acetylene-resistant mutants were isolated from strain DJ939.…”

Section: Methodsmentioning

confidence: 99%

Isolation and Characterization of an Acetylene-resistant Nitrogenase

Christiansen¹,

Cash²,

Seefeldt³

et al. 2000

Journal of Biological Chemistry

111

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A genetic strategy was developed for the isolation of a mutant strain of Azotobacter vinelandii that exhibits in vivo nitrogenase activity resistant to inhibition by acetylene. Examination of the kinetic features of the altered nitrogenase MoFe protein produced by this strain, which has serine substituted for the ␣-subunit Gly 69 residue, is consistent with other studies that indicate the MoFe protein normally contains at least two acetylene binding/reduction sites. The first of these is a high affinity site and is the one primarily accessed during typical acetylene reduction assays. Results of the present work indicate that this acetylene binding/reduction site is not directly relevant to the mechanism of nitrogen reduction because it can be eliminated or severely altered without significantly affecting nitrogen reduction. Elimination of this site also results in the manifestation of a low affinity acetylene-binding site to which both acetylene and nitrogen are able to bind with approximately the same affinity. In contrast to the normal enzyme, nitrogen and acetylene binding to the altered MoFe protein are mutually competitive. The location of the ␣-Ser 69 substitution is interpreted to indicate that the 4Fe-4S face of the FeMo cofactor capped by the ␣-subunit Val 70 residue is the most likely region within FeMo cofactor to which acetylene binds with high affinity.

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“…Plasmids used in this work are not capable of autonomous replication in A. vinelandii, and they are listed in Table I. A. vinelandii cells were cultured in a defined minimal medium without the addition of a fixed nitrogen source (24).…”

Section: Plasmids and Strainsmentioning

confidence: 99%

Iron-Sulfur Cluster Assembly

Santos

Smith

Frazzon

et al. 2004

Journal of Biological Chemistry

113

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The NifU protein is a homodimer that is proposed to provide a molecular scaffold for the assembly of The Azotobacter vinelandii NifU protein is an ϳ60-kDa homodimer proposed to provide a molecular scaffold for formation of [Fe-S] clusters or Fe-S cluster precursors required for full activation of the nitrogenase catalytic components (1, 2). Primary sequence comparisons among NifU homologs indicate that it is a modular protein having three distinct domains (3,4). The N-terminal domain includes three cysteine residues conserved among all known NifU and NifU-like proteins. Incubation of the recombinantly expressed NifU N-terminal domain with NifS (a cysteine desulfurase), L-cysteine, and Fe 2ϩ results in formation of labile [2Fe-2S] clusters on the NifU fragment, providing evidence that this domain could provide a scaffold for assembly of "transient" [Fe-S] clusters destined for nitrogenase activation (2). In support of this hypothesis biochemical and genetic studies established that individual substitution of any of the three cysteine residues contained in the N-terminal domain of NifU impaired but did not eliminate the physiological maturation of the nitrogenase component proteins (3).It was subsequently shown that the N-terminal NifU domain is highly homologous to a small protein designated IscU (5). IscU also contains three conserved cysteines and can provide a scaffold for the sequential in vitro assembly of [2Fe-2S] and [4Fe-4S] clusters when incubated with IscS (a NifS cysteine desulfurase homolog), L-cysteine, and Fe 2ϩ (6, 7). The IscS and IscU proteins together with a suite of other proteins, IscA, HscB, HscA, and Fdx, whose corresponding genes are clustered on both the A. vinelandii and Escherichia coli genomes, have been shown by genetic experiments using E. coli to be involved in the maturation of a variety of Fe-S cluster-containing proteins involved in intermediary metabolism, such as aconitase and glutamate synthase (8 -11). In the case of A. vinelandii, the iscSUAhscBAfdx gene cluster cannot be deleted because this gene cluster is essential under growth conditions for which the nif-specific genes are either expressed or not expressed (5). Thus, under nitrogen fixing conditions NifU is apparently unable to effectively substitute for IscU function. Similarly, NifU deletion strains are severely impaired in their capacity for diazotrophic growth, indicating that IscU cannot effectively replace the function of NifU (12). In contrast, the iscSUAhscBAfdx genes are not essential in E. coli because there is some redundant function provided by another [Fe-S] cluster assembly apparatus encoded by sufABCDSE. Elegant genetic studies have demonstrated that the E. coli iscSUAhscBAfdx genes are essential in the absence of sufABCDS and vice versa (13). Although the A. vinelandii genome encodes proteins homologous to SufSE, recently shown to encode a two-component cysteine desulfurase (14 -16), it clearly does not contain homologs to sufBCD, which probably explains why the "isc" gene cluster is essential in this o...

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Formation of the nitrogen-fixing enzyme system in Azotobacter vinelandii

Cited by 293 publications

References 5 publications

Effects of Oxygen on Acetylene Reduction, Cytochrome Content and Respiratory Activity of Azotobacter chroococcum

Effects of Oxygen on Acetylene Reduction, Cytochrome Content and Respiratory Activity of Azotobacter chroococcum

Isolation and Characterization of an Acetylene-resistant Nitrogenase

Iron-Sulfur Cluster Assembly

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