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The activity of the nitrogenase enzyme in the diazotroph Azospirillum brasilense is reversibly inactivated by ammonium through ADP-ribosylation of the nitrogenase NifH subunit. This process is catalyzed by DraT and is reversed by DraG, and the activities of both enzymes are regulated according to the levels of ammonium through direct interactions with the PII proteins G1nB and G1nZ. We have previously shown that DraG interacts with G1nZ both in vivo and in vitro and that DraT inter- acts with G1nB in vivo. We have now characterized the influence of PII uridylylation status and the PII effectors (ATP, ADP, and 2-oxoglutarate) on the in vitro formation of DraT-G1nB and DraG-G1nZ complexes. We observed that both interactions are maximized when PII proteins are deuridylylated and when ADP is present. The DraT-G1nB complex formed in vivo was purified to homogeneity in the presence of ADP. The stoichiometry of the DraT-G1nB complex was determined by three independent approaches, all of which indicated a 1:1 stoichiometry (DraT monomer:GlnB trimer). Our results suggest that the intracellular fluctuation of the PII ligands ATP, ADP, and 2-oxoglutarate play a key role in the post-translational regulation of nitrogenase activity. [ABSTRACT FROM AUTHOR]