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

Abstract

Using directed mutagenesis, amino acid substitutions have been made in the alpha- and beta-subunits of the klebsiella pneumoniae nitrogenase component 1 at positions normally occupied by conserved cysteine or tyrosine residues. Nif+, Nif- and intermediate phenotypes have been obtained. To extend our earlier biochemical characterization (Kent et al., 1989) the electrophoretic mobility 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 form found in purified nitrogenase correlated approximately with the specific activity of the extract. In contrast, after electrophoresis, component 1 from Nif- mutants exhibited either a major low-mobility form or a fast-moving form. Analysis of nitrogenase polypeptides synthesized in the absence of co-factor (FeMoco) allowed us to conclude that changing cysteine 275 to alanine in the alpha-subunit produces component 1 defective in its interaction with FeMoco. Substitution of other conserved cysteine residues by alanine appears to prevent early steps in nitrogenase assembly or to promote degradation. Two single mutations (cysteine 89 to alanine in the alpha-subunit and cysteine 94 to alanine in the beta-subunit) which are tightly Nif- can be combined to produce a weakly active nitrogenase, indicating regions involved in the interaction between subunits.

Nitrogenase metalloclusters: structures, organization, and synthesis

“…This conclusion was based on the observation that only apo-MoFe which contained Fe (P clusters) was receptive to FeMo-co, and incorporation of such Fe required the activities of genes postulated to be involved in P cluster assembly (54). This possibility is also substantiated by amino acid substitution studies which have shown that certain altered MoFe proteins having disrupted P cluster environments do not appear to have any associated FeMo-co (9,26).…”

Nucleotide sequence and mutational analysis of the vnfENX region of Azotobacter vinelandii

“…There are five Cys residues in the NifD protein and three in the NifK protein that are conserved across nitrogenase proteins from a wide variety of nitrogen-fixing organisms (9,14); some of these Cys residues are thought to form ligands with either FeMoco or P clusters (15,16,28). Four of the five Cys residues in NifD and one of the three Cys residues in NifK are conserved in NifE and NifN, respectively.…”

Isolation and characterization of nitrogenase MoFe protein from the mutant strain pHK17 of Klebsiella pneumoniae in which the two bridging cysteine residues of the P-clusters are replaced by the non-coordinating amino acid alanine

“…On the basis of the Mo contents and EPR spectroscopy of the mutant fractions, we propose that the loss of the P-clusters causes (i) the physical loss or inhibition of binding of some FeMoco ; (ii) the EPR and catalytic inactivation of some FeMoco ; and\or (iii) the incorporation of a FeMoco-like species into the FeMoco site of the mutant molecules. X-ray crystallographic structures of the MoFe protein [4] showed that the FeMoco clusters are bound solely to the αsubunit and that the P-clusters, as deduced from earlier sitedirected mutagenesis experiments [5], are bound at the interface of the αβ pairs of subunits. FeMoco can be considered as consisting of 4Fe-3S and 1Mo-3Fe-3S clusters which are bridged by three inorganic sulphur atoms (Figure 1, left panel).…”

“…In order to understand the structural and functional roles and relationships of FeMoco and P-clusters in nitrogenase, systematic site-directed mutagenesis programmes have been undertaken. Both conserved and non-conserved cysteine residues of the MoFe proteins of K. pneumoniae [5,11] and Azotobacter inelandii [12][13][14][15][16][17] have been replaced by other residues. One of the mutants of K. pneumoniae, pHK17, prepared in this laboratory, is unique.…”

“…One of the mutants of K. pneumoniae, pHK17, prepared in this laboratory, is unique. It carries substitutions in Kp1 of the two bridging cysteine residues α-Cys-89 and β-Cys-94 at the P-cluster site (Figure 1, right panel) by a non-co-ordinating residue, alanine [5]. From the Figure it appears that this replacement is likely to result in complete breakdown of the P-clusters.…”