Structural basis for VO2+-inhibition of nitrogenase activity: (B) pH-sensitive inner-sphere rearrangements in the 1H-environment of the metal coordination site of the nitrogenase Fe–protein identified by ENDOR spectroscopy

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.

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Synthesis of Novel Carbazole Dendrimers Having a Metal Coordination Site

Chemistry Letters ◽

10.1246/cl.2003.674 ◽

2003 ◽

Vol 32 (8) ◽

pp. 674-675 ◽

Cited By ~ 6

Author(s):

Atsushi Kimoto ◽

Jun-Sang Cho ◽

Masayoshi Higuchi ◽

Kimihisa Yamamoto

Keyword(s):

Metal Coordination ◽

Coordination Site

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

Microbiology ◽

10.1099/mic.0.045831-0 ◽

2011 ◽

Vol 157 (6) ◽

pp. 1834-1840 ◽

Cited By ~ 4

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.

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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

Biochemical Journal ◽

10.1042/bj3260637 ◽

1997 ◽

Vol 326 (3) ◽

pp. 637-640 ◽

Cited By ~ 4

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.

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

Biochemical Journal ◽

10.1042/bj2560429 ◽

1988 ◽

Vol 256 (2) ◽

pp. 429-432 ◽

Cited By ~ 69

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.

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