Site-directed mutagenesis of the Klebsiella pneumoniae nitrogenase. Effects of modifying conserved cysteine residues in the alpha- and beta-subunits.
The five conserved cysteine residues present in the alpha-subunit and the three conserved cysteine residues present in the beta-subunit of nitrogenase component 1 were individually changed to alanine. Mutations in the alpha-subunit at positions 63, 89, 155 and 275 and in the beta-subunit at positions 69, 94 and 152 all resulted in a loss of diazotrophic growth and component 1 activity and loss of the normal e.p.r. signal of the component 1 protein. Component 2 activity was retained. Replacement of cysteine-184 in the alpha-subunit with alanine greatly diminished, but did not eliminate, diazotrophic growth and component 1 activity. Substitution of serine for cysteine at position 152 in the beta-subunit, in contrast with the substitution of alanine at this position, resulted in the formation of active component 1. Replacement of the non-conserved cysteine-112 in the beta-subunit with alanine did not greatly perturb diazotrophic growth or the activity of component 1. Extracts prepared from a mutant, with cysteine-275 of the alpha-subunit replaced by alanine, complemented extracts of a mutant unable to synthesize the iron-molybdenum cofactor of nitrogenase, indicating that the alanine-275 substitution increases the availability of cofactor. Furthermore extracts of this mutant exhibited an e.p.r. signal similar to that of extracted iron-molybdenum cofactor. These data suggest a role for cysteine-275 as a ligand to the cofactor.
Kent
H M
HM
A.F.R.C. Institute of Plant Science Research, University of Sussex, Brighton, U.K.
Ioannidis
I
I
Gormal
C
C
Smith
B E
BE
Buck
M
M
D000114:Acetylene / Q000378:metabolism; D003001:Cloning, Molecular; D003545:Cysteine; D004252:DNA Mutational Analysis; D004578:Electron Spin Resonance Spectroscopy; D005798:Genes, Bacterial; D005816:Genetic Complementation Test; D007711:Klebsiella pneumoniae / Q000235:genetics*; D009586:Nitrogen Fixation / Q000235:genetics*; D009591:Nitrogenase / Q000502:physiology*; D011994:Recombinant Proteins; D013329:Structure-Activity Relationship
D011994:Recombinant Proteins; D009591:Nitrogenase; D003545:Cysteine; D000114:Acetylene