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 alpha and beta subunits of the MoFe protein of nitrogenase were analyzed. When synthesized without its partner, the beta subunit accumulated as a soluble beta 4 tetramer. In contrast, when the alpha 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 beta subunit which contained a large internal deletion, such that the alpha subunit could participate in the assembly of small amounts of an alpha 2 beta 2 holoprotein. When synthesized alone, the beta subunit was remarkably stable, even when the protein contained a large internal deletion. The alpha subunit, however, was much more rapidly degraded than the beta subunit, both when it was synthesized alone in its native background and when it was synthesized with its beta subunit partner in a foreign background. Antibodies raised against purified alpha 2 beta 2 MoFe protein recognized epitopes only on the nondenatured beta subunit and not on the nondenatured alpha subunit. Our findings that all epitopes for the alpha2beta2 tetramer appeared to be on the beta subunit, that the beta subunit assembled into beta4 tetramers, and that the alpha subunit alone was very insoluble, combined with the previous finding that the Fe protein binds to the beta 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 beta subunit has a more surface location than the alpha subunit in the alpha2beta2 tetramer.
Li
J G
JG
Department of Molecular Biology and Biochemistry, University of California, Irvine 92717.
Tal
S
S
Robinson
A C
AC
Dang
V
V
Burgess
B K
BK
D000906:Antibodies; D001395:Azotobacter / Q000235:genetics*; D002872:Chromosome Deletion; D003429:Cross Reactions; D005798:Genes, Bacterial; D005779:Immunodiffusion; D046911:Macromolecular Substances; D008983:Molybdoferredoxin / Q000032:analysis / Q000235:genetics* / Q000302:isolation & purification; D009586:Nitrogen Fixation / Q000235:genetics*
D000906:Antibodies; D046911:Macromolecular Substances; D008983:Molybdoferredoxin