Altered nitrogenase MoFe proteins from Azotobacter vinelandii. Analysis of MoFe proteins having amino acid substitutions for the conserved cysteine residues within the beta-subunit

Abstract

The regions surrounding the three strictly conserved cysteine residues (positions 70, 95 and 153) in the beta-subunit of the Azotobacter vinelandii nitrogenase MoFe protein have been proposed to provide P-cluster environments [Dean, Setterquist, Brigle, Scott, Laird & Newton (1990) Mol. Microbiol. 4, 1505-1512]. In the present study, each of these cysteine residues was individually substituted by either serine or alanine by site-directed mutagenesis of the nifK gene, which encodes the MoFe protein beta-subunit. A mutant strain for which the codon for Cys-153 is removed was also isolated. Significant structural or functional roles are indicated for the cysteine residues at positions 70 and 95, where substitution by either serine or alanine eliminates diazotrophic growth of the resulting strains and abolishes or markedly decreases both MoFe-protein acetylene-reduction activity and the intensity of the whole-cell S = 3/2 e.p.r. signal. Changes introduced at position 153 have various effects on the functional properties of the enzyme. The strains produced either by deletion of the Cys-153 residue or its substitution by serine exhibit only a moderate decrease in diazotrophic growth and MoFe-protein activity and no loss of the whole-cell e.p.r.-signal intensity. In contrast, substitution by alanine eliminates diazotrophic growth and very markedly decreases both MoFe-protein activity and e.p.r.-signal intensity. These results are interpreted in terms of a metallocluster-driven protein rearrangement. After purification of the altered MoFe protein, in which serine replaces Cys-153, an investigation of its catalytic and spectroscopic properties confirms that neither the FeMo cofactor, i.e. the substrate-reduction site, nor the component-protein interaction site has been affected. Instead, these data indicate a disruption in electron transfer within the MoFe protein, which is consistent with a role for this residue (and region) at the P clusters.