The nitrogenase components of Anabaena variabilis were isolated to prepare a specific antiserum against the cyanobacterial Fe protein. Thereby, the status of the Fe protein subunits could be analyzed after in‐vivo inactivation of nitrogenase. Firstly, nitrogenase activity was switched off by adding ammonia to intact filaments accompanied with the appearance of a slower migrating subunit of the Fe protein as shown by immunospecific Western blots. Inactivation of nitrogenase was correlated with about equal amounts of modified and non‐modified subunits of the Fe protein. Secondly, a rapid inactivation of nitrogenase was achieved by treatment of cells with oxygen, which was followed by a slow appearance of the modified subunit of the Fe protein.

In the heterocystous cyanobacterium Anabaena variabilis, a change in nitrogenase activity and concomitant modification of dinitrogenase reductase (the Fe protein of nitrogenase) was induced either by NH4Cl at pH 10 (S. Reich and P. Boger, FEMS Microbiol. Lett. 58:81-86, 1989) or by cessation of C supply resulting from darkness, CO2 limitation, or inhibition of photosystem II activity. Modification induced by both C limitation and NH4Cl was efficiently prevented by anaerobic conditions. Under air, endogenously stored glycogen and added fructose protected against modification triggered by C limitation but not by NH4Cl. With stored glycogen present, dark modification took place after inhibition of respiration by KCN. Reactivation of inactivated nitrogenase and concomitant demodification of dinitrogenase reductase occurred after restoration of diazotrophic growth conditions. In previously C-limited cultures, reactivation was also observed in the dark after addition of fructose (heterotrophic growth) and under anaerobiosis upon reillumination in the presence of a photosynthesis inhibitor. The results indicate that modification of dinitrogenase reductase develops as a result of decreased carbohydrate-supported reductant supply of the heterocysts caused by C limitation or by increased diversion of carbohydrates towards ammonia assimilation. Apparently, a product of N assimilation such as glutamine is not necessary for modification. The increase of oxygen concentration in the heterocysts is a plausible consequence of all treatments causing Fe protein modification.In the cyanobacterium Anabaena variabilis, aerobic nitrogen fixation by the enzyme complex nitrogenase (EC 1.18.6.1) is located in the heterocysts (12,13). Heterocysts are devoid of an oxygenic photosystem II and ribulose-1,5-bisphosphate carboxylase, which are operative in vegetative cells. Thus, N2 fixation is spatially separated from water photolysis and coupled ATP formation. The necessary ATP is generated in the heterocysts by photosystem Imediated photophosphorylation and by oxidative phosphorylation. Reductants are produced by dissimilation of fixed carbon imported from vegetative cells into heterocysts (for a review, see reference 41).It is accepted that cyanobacterial nitrogenase activity is controlled at the level of gene expression (13) and by the ability of cells to modulate the supply of reductants and ATP (7,8). Under air, nitrogenase activity in vivo is known to be inhibited by dark treatment (9, 33) and by photosynthesis inhibitors such as 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), which is considered to be due to reductant limitation of heterocysts and concurrent oxygen inactivation of the heterocystous nitrogenase enzyme complex (24, 25). The repressing effect of excess ammonia and other nitrogenous compounds was attributed to enzyme turnover and regulation of enzyme biosynthesis (see, for example, references 4, 13, and 33). Some authors interpreted these effects in terms of inhibition of nitrogenase activity, possibly exerted by glutami...

The nitrogen-fixing cyanobacterium Anabaena uariabilis (ATCC 29413) was cultivated as continuous culture under a 12 h : 12 h light-dark cycle. In the light, photosynthetic activity resulted in a continuous increase in cellular glycogen content, followed by an almost complete dissimilation of the polysaccharide during the dark period. Nitrogenase activity, assayed by the acetylene reduction technique, was low at the end of the dark period and increased quickly upon illumination to reach a maximum after 4 to 6 h of light. The activity rapidly declined after darkening the culture. Increase and decrease of activity were accompanied by a change in the electrophoretic mobility of the Fe-protein of nitrogenase (dinitrogenase reductase) indicative of enzyme modification being involved in the diurnal control of nitrogenase activity. Modification and demodification of the Fe-protein were not coupled to the cell cycle since they followed darkening and illumination when the light or dark periods were changed. Addition of fructose increased nitrogenase activity even in darkness and caused demodification of the Fe-protein. Ammonium chloride supplied at the onset of illumination slowed down the increase of nitrogenase activity. A delayed inhibition of the enzyme was accompanied by partial Feprotein modification only. The reaction was completed after transfer to darkness. The function of enzyme modification in maintaining a constant C : N ratio is discussed and a dominating role of carbohydrate supply in this regulation is indicated by the reported findings.

Cyanobacterium . Anabaena variabilis, Nitrogenase Regulation, Ammonia, Methylamine Using the heterocystous cyanobacterium A nabaena variabilis (ATCC 29413) in an alkaline environment its nitrogenase activity is rapidly inhibited by ammonia and methylamine. Nitro­genase inhibition by ammonia is probably caused by a mechanism comparable to the switch-off effect, which has been described for several species of the Rhodospirillaceae, whereas methyl-amine-induced inhibition is caused by an uncoupling effect only. Evidence for these different effects is obtained by comparing nitrogenase activity in cell-free extracts of filaments pretreated by ammonia or methylamine. In addition, ammonia-dependent nitrogenase inhibition is shown to be dependent on protein synthesis and on light intensity.

Intact filaments of the cyanobacterium Anabaena variabilis switch off nitrogenase activity very rapidly upon addition of NH4Cl when incubated in an alkaline environment (pH 10.0) permitting a fast NH3‐influx into the cells. When assayed in cell‐free extracts (prepared from ammonia‐treated filaments), nitrogenase remains inhibited in the presence of an ATP‐regenerating system. Furthermore, l‐methionine‐d,l‐sulfoximine, an inhibitor of glutamine synthetase, added to the filaments, prevents inactivation of nitrogenase by ammonia, showing that ammonia is not the compound directly responsible for nitrogenase switch‐off.