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.

Nitrogenase MoFe protein (Kp1) from the mutant strain pHK17 or Klebsiella pneumoniae has been purified to give three catalytically active fractions. In this mutant, each of the two bridging cysteine ligands to the P-clusters, alpha-Cys-89 and beta-Cys-94, has been replaced by a non-coordinating residue, alanine. SDS/PAGE and earlier native gels showed that the three fractions retained the normal alpha 2 beta 2 tetrameric form of wild-type Kp1; therefore we conclude that in each of the fractions the subunits are folded differently, thus resulting in different surface charges and allowing separation of the fractions on ion-exchange chronatography. Earlier EPR and magnetic CD data had shown that the mutant fractions contain P-clusters, and thus the mutated residues are not as essential for maintaining the integrity of the P-clusters as they appear from the X-ray structure. The specific activity of each of the three fractions was less than that of wild-type Kp1, the most active fraction having only 50% of wild-type activity. No change in substrate specificity or in the relative distribution of electrons to various substrates was found. The relationship between ATP hydrolysis and substrate-reducing activity, the EPR spectra of the S = 3/2 spin state of the iron-molybdenum cofactor (FeMoco) and the pH profile of acetylene-reduction activities of the three fractions did not differ significantly from those exhibited by wild-type Kp1. The specific activities of the three mutant fractions and of wild-type Kp1 were linearly proportional to the intensity of the S = 3/2 EPR signal from the FeMoco centres. This implies that those molecules of the three mutant fractions and the wild-type protein that contain EPR-active FeMoco are fully active, i.e. that the Cys to Ala substitution of the P-cluster ligands does not affect the specific activity of the protein. This in turn implies that the P-clusters are not directly associated with the rate-limiting step in enzyme turnover. We conclude that the lower specific activities of the mutant fractions are observed because the fractions are mixtures of species containing a full complement of FeMoco and P-clusters and species lacking some or all of these clusters. 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.