ANALYSIS OF AZOTOBACTER-VINELANDII STRAINS CONTAINING DEFINED DELETIONS IN THE NIFD AND NIFK GENES

Strains of Azotobacter vinelandii which contain defined deletions within the nifD and nifK genes which encode, respectively, the α and β subunits of the MoFe protein of nitrogenase were analyzed. When synthesized without its partner, the β subunit accumulated as a soluble β4 tetramer. In contrast, when the α subunit was present without its partner, it accumulated primarily as an insoluble aggregate. The solubility of this protein was increased by the presence of a form of the β subunit which contained a large internal deletion, such that the α subunit could participate in the assembly of small amounts of an α2β2 holoprotein. When synthesized alone, the β subunit was remarkably stable, even when the protein contained a large internal deletion. The α subunit, however, was much more rapidly degraded than the β subunit, both when it was synthesized alone in its native background and when it was synthesized with its β subunit partner in a foreign background. Antibodies raised against purified α2β2 MoFe protein recognized epitopes only on the nondenatured β subunit and not on the nondenatured β subunit. Our findings that all epitopes for the α2β2 tetramer appeared to be on the β subunit, that the β subunit assembled into β4 tetramers, and that the β subunit alone was very insoluble, combined with the previous finding that the Fe protein binds to the β subunit (A.H. Willing, M.M. Georgiadis, D.C. Rees, and J.B. Howard, J. Biol. Chem. 264:8499-8503, 1989) all suggest that the β subunit has a more surface location than the α subunit in the α2β2 tetramer.