title: Ionic interactions in the nitrogenase complex. Properties of Fe-protein containing substitutions for Arg-100.
abstract: A series of Azotobacter vinelandii strains have been constructed in which the nitrogenase Fe-protein (Av2) was altered by substitutions for Arg-100. This invariant residue is a likely partner in a salt bridge with the MoFe-protein and, in some species, is the site of reversible regulation by ADP-ribosylation (Pope, M. R., Murrell, S. A., and Ludden, P. W. (1985) Proc. Natl. Acad. Sci. U. S. A. 82, 3173-3177). Although we find that arginine is the optimum amino acid, other residues in this position could support diazotrophic growth. These results were surprising because Klebsiella pneumoniae Fe-protein substituted by His-100 had been reported to be inactive (Lowery, R. G., Chang, C. L., Davis, L. C., McKenna, M.-C., Stevens, P. J., and Ludden, P. W. (1989) Biochemistry 28, 1206-1212). Two altered Fe-proteins (Av2-R100Y, the tyrosyl form, and Av2-R100H, the histidyl form) were isolated and, in contrast to this earlier report, we found that both had some activity in acetylene reduction. However, both altered proteins exhibited a decreased maximum velocity (35 and 3% of wild type, respectively) and were strongly inhibited by excess MoFe-protein. These adverse activity parameters were also manifest in the increased sensitivity of the altered proteins to inhibition by salts. Indeed, the salt sensitivity of Av2-R100H is so significant that its activity is masked in the normal assay and is easily missed. In addition, for Av2-R100H, substrate reduction is substantially uncoupled from MgATP hydrolysis. These results suggest that substitutions for Arg-100 may decrease the affinity of the Fe-protein for the MoFe-protein prior to electron transfer but increase affinity after electron transfer. Hence, the role of Arg-100 may be to provide the optimum balance in stabilities of these two complexes for maximum efficiency in substrate reduction.
authors.affiliation: Department of Biochemistry, University of Minnesota, Minneapolis 55455.
authors.forename: D
authors.initials: D
authors.lastname: Wolle
authors.forename: C
authors.initials: C
authors.lastname: Kim
authors.forename: D
authors.initials: D
authors.lastname: Dean
authors.forename: J B
authors.initials: JB
authors.lastname: Howard
mesh_terms: D000114:Acetylene / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D001120:Arginine / Q000378:metabolism*; D016948:Azotobacter vinelandii / Q000235:genetics / Q000378:metabolism*; D002384:Catalysis; D005798:Genes, Bacterial; D007477:Ions; D007711:Klebsiella pneumoniae / Q000378:metabolism; D009154:Mutation; D009591:Nitrogenase / Q000235:genetics / Q000378:metabolism*; D010084:Oxidation-Reduction; D010088:Oxidoreductases; D013379:Substrate Specificity
chemical_list: D007477:Ions; D000255:Adenosine Triphosphate; D001120:Arginine; D010088:Oxidoreductases; D009591:Nitrogenase; C029130:nitrogenase reductase; D000114:Acetylene