Posttranslational regulation of nitrogenase activity by fixed nitrogen in Azotobacter chroococcum.
Using anti-(Fe protein) antibody raised against the Fe protein of the photosynthetic bacterium Rhodospirillum rubrum, it was found that the Fe protein component of nitrogenase (EC 1.18.2.1) from Azotobacter chroococcum cells subjected to an ammonium shock, and hence with an inactive nitrogenase, appeared as a doublet in Western blot analysis of cell extracts. The Fe protein incorporated [32P]phosphate and [3H]adenine in response to ammonium treatment, and L-methionine-DL-sulfoximine, an inhibitor of glutamine synthetase (L-glutamate: ammonia ligase (ADP forming), EC 6.3.1.2), prevented Fe protein from inhibition and radioisotope labelling. These results support that A. chroococcum Fe protein is most likely ADP-ribosylated in response to ammonium. After ammonium treatment, when in vivo activity was completely inhibited, Fe-protein modification was still increasing. This suggests the existence of another mechanism of nitrogenase inhibition faster than Fe-protein modification. When ammonium was intracellularly generated instead of being externally added, as occurs with the short-term nitrate inhibition of nitrogenase activity observed in A. chroococcum cells simultaneously fixing molecular nitrogen and assimilating nitrate, a covalent modification of the Fe protein was likewise demonstrated.
Muñoz-Centeno
M C
MC
Instituto de Bioquímica Vegetal y Fotosíntesis, Universidad de Sevilla-Consejo Superior de Investigaciones Científicas, Facultad de Biología, Spain.
Ruiz
M T
MT
Paneque
A
A
Cejudo
F J
FJ
D000246:Adenosine Diphosphate Ribose / Q000378:metabolism; D000643:Ammonium Chloride / Q000494:pharmacology*; D001395:Azotobacter / Q000187:drug effects / Q000201:enzymology*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005974:Glutamate-Ammonia Ligase / Q000037:antagonists & inhibitors; D007700:Kinetics; D008717:Methionine Sulfoximine / Q000494:pharmacology; D009566:Nitrates / Q000494:pharmacology; D009591:Nitrogenase / Q000037:antagonists & inhibitors* / Q000737:chemistry; D010088:Oxidoreductases; D017680:Potassium Compounds / Q000494:pharmacology; D011499:Protein Processing, Post-Translational / Q000187:drug effects*
D004791:Enzyme Inhibitors; D009566:Nitrates; D017680:Potassium Compounds; D000643:Ammonium Chloride; D008717:Methionine Sulfoximine; D000246:Adenosine Diphosphate Ribose; D010088:Oxidoreductases; D009591:Nitrogenase; C029130:nitrogenase reductase; D005974:Glutamate-Ammonia Ligase; C023844:potassium nitrate