scholarly journals The role of Mg2+ and Mn2+ in the enzyme-catalysed activation of nitrogenase Fe protein from Rhodospirillum rubrum

1983 ◽  
Vol 213 (3) ◽  
pp. 741-749 ◽  
Author(s):  
J H Guth ◽  
R H Burris
Keyword(s):  
Metal Ion ◽  
Rhodospirillum Rubrum ◽  
Nitrogenase Activity ◽  
Optimal Conditions ◽  
Fe Protein ◽  
C2h2 Reduction ◽  
Site Of Action ◽  
A Site ◽  
Free Metal

The activation of the Fe protein of nitrogenase (Rr2) from glutamate-grown Rhodospirillum rubrum by activating enzyme (AE) was investigated. AE is confirmed to have Mr about 20 000 and is shown to operate catalytically. There is a role in activation for metal-ion-ATP, which can be met by either MnATP or MgATP. There is also a site of action for free metal ions. This site prefers Mn2+ (apparent Kd approx. 20 microM) over Mg2+ (apparent Kd approx. 20 mM) by a factor of 1000-fold. Non-activated Rr2 does not contain this binding site. MnATP is an inhibitor of C2H2 reduction, and excess Mg2+ inhibits both AE activity and C2H2 reduction, when each is studied independently under otherwise optimal conditions. The activity of AE is increased in normal reaction mixtures (in which AE activity and nitrogenase activity occur simultaneously) by Mg2+ concentrations in excess of ATP concentrations; this occurs because the excess Mg2+ prevents ATP from chelating the free Mn2+ necessary for optimal AE activity.

scholarly journals Adenine nucleotide levels in Rhodospirillum rubrum during switch-off of whole-cell nitrogenase activity

1984 ◽  
Vol 224 (3) ◽  
pp. 961-969 ◽  
Author(s):  
T D Paul ◽  
P W Ludden
Keyword(s):  
Protein Modification ◽  
Rhodospirillum Rubrum ◽  
Adenine Nucleotide ◽  
Nitrogenase Activity ◽  
Energy Charge ◽  
Whole Cell ◽  
Fe Protein ◽  
Nucleotide Pools ◽  
Phenazine Methosulphate

Adenine nucleotide pools were measured in Rhodospirillum rubrum cultures that contained nitrogenase. The average energy charge [([ATP] + 1/2[ADP])/([ATP] + [ADP] + [AMP])] was found to be 0.66 and 0.62 in glutamate-grown and N-limited cultures respectively. Treatment of glutamate-grown cells with darkness, ammonia, glutamine, carbonyl cyanide m-chlorophenylhydrazone, or phenazine methosulphate resulted in perturbations in the adenine nucleotide pools, and led to loss of whole-cell nitrogenase activity and modification in vivo of the Fe protein. Treatment of N-limited cells resulted in similar changes in adenine nucleotide pools but not enzyme modification. No correlations were found between changes in adenine nucleotide pools or ratios of these pools and switch-off of nitrogenase activity by Fe protein modification in vivo. Phenazine methosulphate inhibited whole-cell activity at low concentrations. The effect on nitrogenase activity was apparently independent of Fe protein modification.

Purification and Mn2+ activation of Rhodospirillum rubrum nitrogenase activating enzyme

1982 ◽  
Vol 152 (2) ◽  
pp. 714-721
Author(s):  
J W Gotto ◽  
D C Yoch
Keyword(s):  
Metal Binding ◽  
Metal Ion ◽  
Rhodospirillum Rubrum ◽  
Gradient Centrifugation ◽  
Sucrose Density Gradient ◽  
Maximum Activity ◽  
Sucrose Density Gradient Centrifugation ◽  
Fe Protein ◽  
Single Polypeptide ◽  
Sparing Effect

The Fe protein activating enzyme for Rhodospirillum rubrum nitrogenase was purified to approximately 90% homogeneity, using DE52-cellulose chromatography and sucrose density gradient centrifugation. Activating enzyme consists of a single polypeptide of molecular weight approximately 24,000. ATP was required for catalytic activity, but was relatively ineffective in the absence of Mg2+. When the concentration of MgATP2- was held in excess, there was an additional requirement for a free divalent metal ion (Mn2+) for enzyme activity. Kinetic experiments showed that the presence of Mg2+ influenced the apparent binding of Mn2+ by the enzyme, resulting in a lowering of the concentration of Mn2+ required to give half-maximum activity (K alpha) as the free Mg2+ concentration was increased. A low concentration of Mn2+ had a sparing effect on the requirement for free Mg2+. There is apparently a single metal-binding site on activating enzyme which preferentially binds Mn2+ as a positive effector, and free Mg2+ can compete for this site.

scholarly journals Role of the Dinitrogenase Reductase Arginine 101 Residue in Dinitrogenase Reductase ADP-Ribosyltransferase Binding, NAD Binding, and Cleavage

2001 ◽  
Vol 183 (1) ◽  
pp. 250-256 ◽  
Author(s):  
Yan Ma ◽  
Paul W. Ludden
Keyword(s):  
Rhodospirillum Rubrum ◽  
Nitrogenase Activity ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Adp Ribosylation ◽  
Dinitrogenase Reductase ◽  
Adp Ribosyltransferase

ABSTRACT Dinitrogenase reductase is posttranslationally regulated by dinitrogenase reductase ADP-ribosyltransferase (DRAT) via ADP-ribosylation of the arginine 101 residue in some bacteria.Rhodospirillum rubrum strains in which the arginine 101 of dinitrogenase reductase was replaced by tyrosine, phenylalanine, or leucine were constructed by site-directed mutagenesis of thenifH gene. The strain containing the R101F form of dinitrogenase reductase retains 91%, the strain containing the R101Y form retains 72%, and the strain containing the R101L form retains only 28% of in vivo nitrogenase activity of the strain containing the dinitrogenase reductase with arginine at position 101. In vivo acetylene reduction assays, immunoblotting with anti-dinitrogenase reductase antibody, and [adenylate-32P]NAD labeling experiments showed that no switch-off of nitrogenase activity occurred in any of the three mutants and no ADP-ribosylation of altered dinitrogenase reductases occurred either in vivo or in vitro. Altered dinitrogenase reductases from strains UR629 (R101Y) and UR630 (R101F) were purified to homogeneity. The R101F and R101Y forms of dinitrogenase reductase were able to form a complex with DRAT that could be chemically cross-linked by 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide. The R101F form of dinitrogenase reductase and DRAT together were not able to cleave NAD. This suggests that arginine 101 is not critical for the binding of DRAT to dinitrogenase reductase but that the availability of arginine 101 is important for NAD cleavage. Both DRAT and dinitrogenase reductase can be labeled by [carbonyl-14C]NAD individually upon UV irradiation, but most 14C label is incorporated into DRAT when both proteins are present. The ability of R101F dinitrogenase reductase to be labeled by [carbonyl-14C]NAD suggested that Arg 101 is not absolutely required for NAD binding.

Effect of pyruvate on the metabolic regulation of nitrogenase activity in Rhodospirillum rubrum in darkness

Microbiology ◽  
2011 ◽  
Vol 157 (6) ◽  
pp. 1834-1840 ◽  
Author(s):  
Tiago Toscano Selao ◽  
Tomas Edgren ◽  
He Wang ◽  
Agneta Norén ◽  
Stefan Nordlund
Keyword(s):  
Nitrogen Source ◽  
Metabolic Regulation ◽  
Rhodospirillum Rubrum ◽  
De Novo ◽  
Nitrogenase Activity ◽  
Signal Transduction Pathway ◽  
Nitrogen Sources ◽  
Synthetase Activity ◽  
Glutamine Synthetase Activity ◽  
Fe Protein

Rhodospirillum rubrum, a photosynthetic diazotroph, is able to regulate nitrogenase activity in response to environmental factors such as ammonium ions or darkness, the so-called switch-off effect. This is due to reversible modification of the Fe-protein, one of the two components of nitrogenase. The signal transduction pathway(s) in this regulatory mechanism is not fully understood, especially not in response to darkness. We have previously shown that the switch-off response and metabolic state differ between cells grown with dinitrogen or glutamate as the nitrogen source, although both represent poor nitrogen sources. In this study we show that pyruvate affects the response to darkness in cultures grown with glutamate as nitrogen source, leading to a response similar to that in cultures grown with dinitrogen. The effects are related to PII protein uridylylation and glutamine synthetase activity. We also show that pyruvate induces de novo protein synthesis and that inhibition of pyruvate formate-lyase leads to loss of nitrogenase activity in the dark.

Role of the free metal ion species in soluble nickel removal by activated sludge

1984 ◽  
Vol 18 (11) ◽  
pp. 883-886 ◽  
Author(s):  
Jeppe S. Nielsen ◽  
Steve E. Hrudey ◽  
Frederick F. Cantwell
Keyword(s):  
Activated Sludge ◽  
Metal Ion ◽  
Nickel Removal ◽  
Ion Species ◽  
Free Metal ◽  
Free Metal Ion

scholarly journals The role of NAD+ as a signal during nitrogenase switch-off in Rhodospirillum rubrum

1997 ◽  
Vol 322 (3) ◽  
pp. 829-832 ◽  
Author(s):  
Agneta NORÉN ◽  
Abdelhamid SOLIMAN ◽  
Stefan NORDLUND
Keyword(s):  
Metabolic Regulation ◽  
Opposite Effect ◽  
Rhodospirillum Rubrum ◽  
Nitrogenase Activity ◽  
Regulatory System ◽  
Glutamate Synthase ◽  
The Other ◽  
Dinitrogenase Reductase ◽  
Adp Ribosyltransferase

The role of NAD+ in the metabolic regulation of nitrogenase, the ‘switch-off’ effect, in Rhodospirillum rubrum has been studied. We now show that the decrease in nitrogenase activity upon addition of NAD+ to R. rubrum is due to modification of dinitrogenase reductase. There was no effect when NAD+ was added to a mutant of R. rubrumdevoid of dinitrogenase reductase ADP-ribosyltransferase, indicating that NAD+ ‘switch-off’ is an effect of the same regulatory system as ammonium ‘switch-off’. We also show that oxaloacetate and α-ketoglutarate function as ‘switch-off’ effectors. On the other hand β-hydroxybutyrate has the opposite effect by shortening the ‘switch-off’ period. Furthermore, by using an inhibitor of glutamate synthase the role of this enzyme in ‘switch-off’ was investigated. The results are discussed in relation to our proposal that changes in the concentration of NAD+ are involved in initiating ‘switch-off’.

Expression of the activating enzyme and Fe protein of nitrogenase from Rhodospirillum rubrum

1982 ◽  
Vol 152 (2) ◽  
pp. 786-791
Author(s):  
E W Triplett ◽  
J D Wall ◽  
P W Ludden
Keyword(s):  
Carbon Source ◽  
Rhodospirillum Rubrum ◽  
Nitrogenase Activity ◽  
N Sources ◽  
Fe Protein ◽  
N Source ◽  
In Vitro Activation ◽  
High Ammonia ◽  
Cells Cultured

Activating enzyme (AE) is responsible for the in vitro activation of inactive Fe protein of nitrogenase from Rhodospirillum rubrum cells cultured anaerobically with glutamate as the N source. The expression of Fe protein and AE was examined in R. rubrum cultured photosynthetically or aerobically on media containing malate as the carbon source. One of the following N sources was used in each culture: glutamate, glutamine, limiting ammonia, high ammonia, glutamate plus histidine, and high ammonia plus histidine. Chromatophores from every culture exhibited AE activity; activity was highest in glutamate-grown cells. Fe protein was observed by rocket immunoelectrophoresis in cultures with nitrogenase activity. Several Nif-, Gln-, and His- mutants of R. rubrum were assayed for AE activity, nitrogenase activity, and Fe protein. Every mutant expressed AE activity, and Fe protein was observed in those cultures with nitrogenase activity. AE from every preparation was O2 labile, and each O2-denatured AE preparation inhibited activation by active AE.

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