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

Abstract

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 In this mutant, each of the two bridging cysteine ligands to the P-clusters, alpha-Cys-89 and beta-Cys-94, has been replaced by a non-coordinating residue, alanine.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.

Long-range interactions between the Fe protein binding sites of the MoFe protein of nitrogenase

“…Purification of the nitrogenase components Kp1 and Cp2 Kp1 was purified from K. pneumoniae strain M5A1 as described previously [25,26]. The specific activity of Kp1 was 1800 nmol of C 2 H 2 reduced min ±1 mg ±1 .…”

Evidence for the Selective Population of FeMo Cofactor Sites in MoFe Protein and Its Molecular Recognition by the Fe Protein in Transition State Complex Analogues of Nitrogenase

“…The nitrogenase proteins Kp1 and Kp2 were purified from K. pneumoniae to homogeneity as described previously (27,28). In the case of Kp1, this method (27) resolved species containing 1.1 and 1.9 molybdenum atoms per molecule and were shown to be free of any contaminating proteins by SDS gel electrophoresis. The specific activities expressed as nmol of hydrogen evolved/min/mg of protein were as follows: Kp1(1.9 Mo), 2100; Kp1(1.1 Mo), 1100; Kp2, 1399.…”

Characterization of a nitrogenase CrFe protein from a mutant UW3 of Azotobacter vinelandii grown on a Cr-containing medium

“…Yousafzai et al [31] reported that under the condition of 1 of FeMoco to P-cluster, the EPR intensity (g≈4.30) of Kp1 (MoFe protein of K. pneumonia) was finely proportional to its activity and the metal contents assembled in FeMoco and P-cluster. Zhou et al [17] successfully used the method to verify the model of ΔnifZ Av1 with 2 of FeMoco to P-cluster.…”

“…So calculated values of the activity and metal content for CrFe protein and residual MoFe protein in the preparation according to the EPR intensity can be used to make contrast with and test the experimental results. With the calibration of the residual Mo content suggested above, under the condition that possible FeCrco and P-cluster in CrFe protein are similar in structure and quantity to those of FeMoco and P-cluster in MoFe protein, the EPR-based activity and metal content of CrFe protein and MoFe protein in the preparation are obtained using the method described by Yousafzai et al [31] , as listed in Table 4. The calculated results correspond to the experimental ones well.…”