FeMo cofactor synthesis by a nifH mutant with altered MgATP reactivity.
We have characterized a Nif- mutant of Azotobacter vinelandii, designated UW91 (Shah, V. K., Davis, L. C., Gordon, J. K., Orme-Johnson, W. H., and Brill, W. J. (1973) Biochim. Biophys. Acta 292, 246-255). The specific Fe protein mutation giving rise to the Nif- phenotype was shown by DNA sequencing and site-directed mutagenesis to be the substitution of a conserved alanine at position 157 by a serine. The UW91 Fe protein was purified and shown to have a normal [4Fe-4S] cluster and normal MgATP binding activity. The substitution of alanine 157 by serine, however, prevents the MgATP-induced conformational change that occurs for the wild-type Fe protein, prevents MgATP hydrolysis, and prevents productive electron transfer to the MoFe protein. The UW91 Fe protein does bind to the MoFe protein to give a normal cross-linking pattern; however, it does not compete very successfully with wild-type Fe protein in an activity assay. The UW91 MoFe protein was also purified and characterized and shown to be indistinguishable from the wild-type protein. Thus, the substitution of Fe protein residue alanine 157 by serine does not change the Fe protein's ability to function in FeMo cofactor biosynthesis or insertion. This demonstrates that these events do not require the MgATP-induced conformational change, MgATP hydrolysis, or productive electron transfer to the MoFe protein.
Gavini
N
N
Department of Molecular Biology and Biochemistry, University of California, Irvine 92717.
Burgess
B K
BK
D000114:Acetylene / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism*; D000595:Amino Acid Sequence; D016948:Azotobacter vinelandii / Q000235:genetics / Q000378:metabolism*; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D002876:Chromosomes, Bacterial; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000235:genetics / Q000302:isolation & purification; D005798:Genes, Bacterial; D007700:Kinetics; D008969:Molecular Sequence Data; D008983:Molybdoferredoxin / Q000096:biosynthesis* / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D009591:Nitrogenase / Q000235:genetics / Q000378:metabolism*; D010084:Oxidation-Reduction; D010088:Oxidoreductases; D010641:Phenotype; D017386:Sequence Homology, Amino Acid
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D008983:Molybdoferredoxin; D000255:Adenosine Triphosphate; D010088:Oxidoreductases; D009591:Nitrogenase; C029130:nitrogenase reductase; D000114:Acetylene