Role of the Dinitrogenase Reductase Arginine 101 Residue in Dinitrogenase Reductase ADP-Ribosyltransferase Binding, NAD Binding, and Cleavage

Abstract

Dinitrogenase reductase is posttranslationally regulated by dinitrogenase reductase ADP-ribosyltransferase (DRAT) via ADP-ribosylation of the arginine 101 residue in some bacteria. Rhodospirillum rubrum strains in which the arginine 101 of dinitrogenase reductase was replaced by tyrosine, phenylalanine, or leucine were constructed by site-directed mutagenesis of the nifH gene. The strain containing the R101F form of dinitrogenase reductase retains 91%, the strain containing the R101Y form retains 72%, and the strain containing 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-dinitrogenase reductase antibody, and [adenylate-32 P]NAD labeling experiments showed that no switch-off of nitrogenase activity occurred in any of the three mutants and no ADP-ribosylation of altered dinitrogenase reductases occurred either in vivo or in vitro. Altered dinitrogenase reductases from strains UR629 (R101Y) and UR630 (R101F) were purified to homogeneity. The R101F and R101Y forms of dinitrogenase reductase were able to form a complex with DRAT that could be chemically cross-linked by 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide. The R101F form of dinitrogenase reductase and DRAT together were not able to cleave NAD. This suggests that arginine 101 is not critical for the binding of DRAT to dinitrogenase reductase but that the availability of arginine 101 is important for NAD cleavage. Both DRAT and dinitrogenase reductase can be labeled by [carbonyl-14 C]NAD individually upon UV irradiation, but most 14 C label is incorporated into DRAT when both proteins are present. The ability of R101F dinitrogenase reductase to be labeled by [carbonyl-14 C]NAD suggested that Arg 101 is not absolutely required for NAD binding.Various procaryotic microorganisms are capable of fixing nitrogen. The essential enzyme in this reaction is the nitrogenase complex, which catalyzes the reduction of nitrogen to ammonium (4). This complex is composed of two separable metalloenzymes designated dinitrogenase and dinitrogenase reductase. Dinitrogenase reductase transfers electrons from an electron donor to dinitrogenase, which then can catalyze the reduction of nitrogen to ammonium. Because nitrogen fixation is an energy-demanding process, it is tightly regulated in response to a number of environmental factors. In addition to transcriptional regulation, a posttranslational nitrogenase regulatory mechanism also exists in certain organisms, such as Rhodospirillum rubrum (33,35). R. rubrum is a free-living, purple, nonsulfur photosynthetic bacterium that fixes nitrogen (20). The activity of its nitrogenase is regulated by the reversible ADP-ribosylation of the arginine 101 of the dinitrogenase reductase (31, 42). Dinitrogenase reductase ADP-ribosyltransferase (DRAT) can transfer ADP-ribose from NAD to dinitrogenase reductase in response to darkness or the presence of ammonium. This ADP-ribosylation ...

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