Analysis of site-directed mutations in the α-and β-subunits of Klebsiella pneumoniae nitrogenase.

Using directed mutagenesis, amino acid substitutions have been made in the α- and β-subunits of the Klebsiella pneumoniae nitrogenase component 1 at positions normally occupied by conserves cysteine or tyrosine residues. Nif', Nif and intermediate pheno-types have been obtained. To extend our earlier biochemical characterization (Kent et al, 1989) the electrophoretic mobiliy of component 1 of the mutant and wild-type nitrogenases has been analysed by non-denaturing gel electrophoresis. The major and minor forms of component 1 separated by this methodology have been probed for by using both polyclonal and monoclonal antibodies. All Nif mutants exhibited a distribution of electrophoretic forms of component 1 comparable to the wild type, and the abundance of the major from found in purified nitrogennase correlated approximately with the specific activity of the extract. In contrast, after electrophoresis, component 1 from Nif mutants exhibited either a major low-mobility from or a fast-moving from. Analysis of co-factor (FeMoco) allowed us to conclude that changing cysteine 275 to alanine in the α-submit produces component 1 defective in its interaction with FeMoco. Substitution of other con-served cysteine residues by alanine appears to prevent early steps in nitrogebnase assembly or to promote degradation. Two single mutations (cysteine89 to alanine in the β-subunit) which are tightly Nif can be combined to produce a weakly active nitrogenase, indicating regions involved in the interaction between subunits.