Inhibition of nitrogenase activity by NH+4 in Rhodospirillum rubrum

Abstract

Nitrogenase activities and the patterns of in vivo inhibition of nitrogenase by NW were compared in Rhodospiilum rubrum grown under several conditions of nitrogen availability. In cells grown on N2 or glutamate plus N2, nitrogenase activity was relatively low and was totally inhibited by added NH: in 15 to 20 min. In contrast, cells grown on glutamate alone displayed higher nitrogenase activity, and NH: had very little effect. Cells grown on limiting amounts of NH: had lower nitrogenase activity, but NH: produced little inhibitory effect. Uptake of NW could be demonstrated under all of these conditions, and this uptake was blocked by DL-methionine-dl-sulfoximine. The data indicated that cells not recently exposed to NHW had no mechanism for rapidly turning off nitrogenase activity in response to sudden additions of NW. In contrast, cells grown in the presence of N2, which form NW internally, inhibited nitrogenase activity relatively quickly in response to added NW.All species of purple nonsulfur photosynthetic bacteria that have been tested can reduce N2 to ammonia. The nitrogenase systems of these bacteria appear unique because the enzyme activity can be reversibly inhibited by ammonium ions. This effect has been observed only in vivo, and it is probably mediated by products of NW assimilation rather than by NW directly (see reference 13 for review). It also has been shown that cell-free extracts from Rhodospirillum rubrum (6,9), Rhodopseudomonas palustris (16), and Rhodopseudomonas capsulata (14) grown with glutamate or N2 as nitrogen sources have low nitrogenase activities, even though the activities of the intact cells were high at the time of harvest. In all cases, these low in vitro activities could be increased in the presence of Mn2+ and Mg-ATP by the addition of a component isolated from the respective chromatophores. This component, designated activating factor, appears to catalyze the release of a small, covalently bound molecule from the iron protein during the activation process (7). The in vitro activation phenomenon might be related to the regulation by ammonium salts observed in vivo, but no in vitro effect of NW on nitrogenase activity has been demonstrated.

Nitrogenase from the photosynthetic bacterium Rhodopseudomonas capsulata: purification and molecular properties.

“…After being switched off, in vitro nitrogenase activity was predominantly in the N2ase R form. These results are in conflict with a recent report that cultures not recently exposed to NH4' have no mechanism for rapidly turning off nitrogenase activity in response to sudden additions of NH4' (26). In the work reported here, nitrogenase activity was specifically assayed by using acetylene reduction, rather than by following H2 evolution (26), which is ambiguous due to possible hydrogen consumption by the uptake hy-drogenase.…”

“…These results are in conflict with a recent report that cultures not recently exposed to NH4' have no mechanism for rapidly turning off nitrogenase activity in response to sudden additions of NH4' (26). In the work reported here, nitrogenase activity was specifically assayed by using acetylene reduction, rather than by following H2 evolution (26), which is ambiguous due to possible hydrogen consumption by the uptake hy-drogenase. Extrapolation of the period of inhibition by various concentrations of NH4' showed that, for nitrogen-starved cultures, added ammonium must be greater than 50 FM to have an inhibitory effect.…”

“…Extrapolation of the period of inhibition by various concentrations of NH4' showed that, for nitrogen-starved cultures, added ammonium must be greater than 50 FM to have an inhibitory effect. This is higher than the concentration range used by Sweet and Burris (26) thus at least partially explaining why they failed to find an inhibitory effect. This minimal concentration (50 ,uM) is much higher than that found for glutamate-grown (10 ,uM) (19) or N2-grown (5 ,uM) (26) cultures.…”

“…This is higher than the concentration range used by Sweet and Burris (26) thus at least partially explaining why they failed to find an inhibitory effect. This minimal concentration (50 ,uM) is much higher than that found for glutamate-grown (10 ,uM) (19) or N2-grown (5 ,uM) (26) cultures. This may reflect a lowered concentration, in nitrogen-starved cells, of the compound (presumably nitrogenous) that triggers the switch-off effect.…”

Inhibition of nitrogenase activity by ammonium chloride in Azotobacter vinelandii

“…This so-called short-term effect is observed in Azotobacter (5), Rhodospirillum (13), Rhodopseudomonas (8,21) and Anabaena (20) species. For the phototrophic organisms, inhibition is dependent on the growth conditions (1,18,20). In organisms that fix nitrogen only under fermentative conditions, like Clostridium and Klebsiella species, no shortterm effect of ammonia is found (2,19).…”