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. 1972 Jul;128(3):655–675. doi: 10.1042/bj1280655

Nitrogenase of Klebsiella pneumoniae. Purification and properties of the component proteins

R R Eady 1, B E Smith 1, K A Cook 1,*, J R Postgate 1
PMCID: PMC1173817  PMID: 4344006

Abstract

1. Nitrogenase from the facultative anaerobe Klebsiella pneumoniae was resolved into two protein components resembling those obtained from other nitrogen-fixing bacteria. 2. Both proteins were purified to homogeneity as shown by the criteria of disc electrophoresis and ultracentrifugal analysis. 3. The larger component had a mol.wt. of 218000 and contained one Mo atom, 17Fe atoms and 17 acid-labile sulphide groups/mol; it contained two types of subunit, present in equal amounts, of mol.wts. 50000 and 60000. All the common amino acids were present, with a predominance of acidic residues. The apparent partial specific volume was 0.73; ultracentrifugal analysis gave s020,w=11.0S and D020,w=4.94×10−7cm2/s. The specific activities (nmol of product formed/min per mg of protein) when assayed with the second nitrogenase component were 1500 for H2 evolution, 380 for N2 reduction, 1200 for acetylene reduction and 5400 for ATP hydrolysis. The reduced protein showed electron-paramagnetic-resonance signals at g=4.3, 3.7 and 2.015; the Mössbauer spectrum of the reduced protein consisted of at least three doublets. The u.v. spectra of the oxidized and reduced proteins were identical. On oxidation the absorbance increased generally throughout the visible region and a shoulder at 430nm appeared. The circular-dichroism spectra of both the oxidized and reduced proteins were the same, consisting mainly of a negative trough at 220nm. 4. The smaller component had mol.wt. 66800 and contained four Fe atoms and four acid-labile sulphide groups in a molecule comprising two subunits each of mol.wt. 34600. All common amino acids except tryptophan were present, with a predominance of acidic residues. The apparent partial specific volume calculated from the amino acid analysis was 0.732, which was significantly higher than that obtained from density measurements (0.69); ultracentrifugal analysis gave s020,w=4.8S and D020,w=5.55×10−7cm2/s. The specific activities (nmol of product formed/min per mg of protein) were 1050 for H2 evolution, 275 for N2 reduction, 980 for acetylene reduction and 4350 for ATP hydrolysis. The protein was not cold-labile. The reduced protein showed electron-paramagnetic-resonance signals in the g=1.94 region. The Mössbauer spectrum of the reduced protein consisted of a doublet at 77°K. The u.v. spectra of reduced and O2-inactivated proteins were identical, and inactivation by O2 generally increased the absorbance in the visible region and resulted in a shoulder at 460nm. The circular-dichroism spectra exhibited a negative trough at 220nm and inactivation by O2 decreased the depth of the trough. 5. The reduction of N2 and acetylene, and H2 evolution, were maximal at a 1:1 molar ratio of the Fe-containing protein to the Mo–Fe-containing protein; excess of the Mo–Fe-containing protein was inhibitory. All reductions were accompanied by H2 evolution. The combined proteins had no ATP-independent hydrogenase activity.

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Selected References

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