Role of the dinitrogenase reductase arginine 101 residue in dinitrogenase reductase ADP-ribosyltransferase binding, NAD binding, and cleavage

Dinitrogenase reductase is posttranslationally regulated by dinitrogenase r eductase ADP-ribosyltransferase (DRAT) via ADP-ribosylation of the arginine 101 residue in some bacteria. Rhodospirillum rubrum strains in which the a rginine 101 of dinitrogenase reductase was replaced by tyrosine, phenylalan ine, or leucine were constructed by site-directed mutagenesis of the nifH g ene. The strain containing the R101F form of dinitrogenase reductase retain s 91%, the strain containing the R101Y form retains 72%, and the strain con taining the R101L form retains only 28% of in vivo nitrogenase activity of the strain containing the dinitrogenase reductase with arginine at position 101, In vivo acetylene reduction assays, immunoblotting with anti-dinitrog enase reductase antibody, and [adenylate-P-32]NAD labeling experiments show ed that no switch-off of nitrogenase activity occurred in any of the three mutants and no ADP-ribosylation of altered dinitrogenase reductases occurre d either in vivo or in vitro. Altered dinitrogenase reductases from strains UR629 (R101Y) and UR630 (R101F) were purified to homogeneity. The R101F an d R101Y forms of dinitrogenase reductase were able to form a complex with B RAT that could be chemically cross-linked by 1-ethyl-3-(3-dimethylaminoprop yl)carbodiimide. The R101F form of dinitrogenase reductase and BRAT togethe r were not able to cleave NAD, This suggests that arginine 101 is not criti cal for the binding of DRAT to dinitrogenase reductase but that the availab ility of arginine 101 is important for NAD cleavage. Both BRAT and dinitrog enase reductase can be labeled by [carbonyl-C-14]NAD individually upon UV i rradiation, but most C-14 label is incorporated into BRAT when both protein s are present. The ability of R101F dinitrogenase reductase to be labeled b y [carbonyl-C-14]NAD suggested that Arg 101 is not absolutely required for NAD binding.