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.

FIG. 1

Immunoblot of crude extracts of strains UR628, UR629, UR630, and UR631 developed with anti-dinitrogenase reductase antibody. Samples were taken before and after a 60-min dark (A) or 90-min ammonium (B) treatment.

FIG. 2

Immunoblot with anti-DRAT antibody, showing the nucleotide dependence of DRAT-dinitrogenase reductase EDC-dependent cross-linking. Cross-linking reactions were performed as described in Materials and Methods except that the following proteins and nucleotides (1.25 mM MgADP, 2 mM NAD) were added to the reaction: lane 1, only DRAT; lane 2, no DRAT; lanes 3 and 4, DRAT and wild-type dinitrogenase reductase; lanes 5 and 6, DRAT and R101Y dinitrogenase reductase; lanes 7 and 8, DRAT and R101F dinitrogenase reductase. Lanes 3, 5, and 7, no NAD; lanes 4, 6, and 8, NAD. All reactions contained 1.25 mM MgADP.

FIG. 3

Phosphorimage of a thin-layer chromatogram showing [¹⁴C]nicotinamide release from [carbonyl-¹⁴C]NAD. Reactions of different dinitrogenase reductases with DRAT were performed as described in Materials and Methods except that different proteins were added to the reaction as follows: lane 1, DRAT only; lane 2, DRAT plus A. vinelandii dinitrogenase reductase; lane 3, DRAT plus wild-type R. rubrum dinitrogenase reductase; lane 4, DRAT plus R101F dinitrogenase reductase.

FIG. 4

Coomassie blue-stained SDS-PAGE gel (A) and its phosphorimage (B) showing proteins labeled upon UV irradiation in the presence of [carbonyl-¹⁴C]NAD. They include chicken egg albumin (lanes 1), cholera toxin A (lanes 2), DRAT (lanes 3), dinitrogenase reductase (DR) (lanes 4), DRAT and dinitrogenase reductase (lanes 5), R101F dinitrogenase reductase (lanes 6), DRAT and R101F dinitrogenase reductase (lanes 7), oxygen-denatured dinitrogenase reductase (lanes 8), and oxygen-denatured R101F dinitrogenase reductase (lanes 9). After reactions were performed as described in Materials and Methods, except with the above-noted proteins added, the SDS-PAGE gel was Coomassie blue stained and exposed to a Molecular Dynamics storage phosphor screen.