The vanadium nitrogenase of Azotobacter chroococcum. Purification and properties of the Fe protein.
1. Nitrogenase activity of a strain of Azotobacter chroococcum lacking the structural genes of Monitrogenase (nifHDK) was associated with a V + Fe-containing protein and an Fe-containing protein [Robson, Eady, Richardson, Miller, Hawkins & Postgate (1986) Nature (London) 322, 388-390; Eady, Robson, Richardson, Miller & Hawkins (1987) Biochem. J. 244, 197-207]. 2. The Fe protein was purified to homogeneity by the criterion of Coomassie Blue staining after electrophoresis in 10% or 17% (w/v) polyacrylamide gels in the presence of SDS. One type of subunit, of Mr 32,000 +/- 2000, was found. 3. The native protein had an Mr of 62,500 +/- 2500 and contained approximately 4 Fe atoms and 4 acid-labile sulphide groups per molecule. The amino acid composition was similar to those of other purified Fe proteins, and, characteristically, tryptophan was absent. The specific activities (nmol of protein/min per mg of protein) when assayed under optimum conditions with the VFe protein from this strain were 1211 for H2 evolution under Ar, 337 for NH3 from N2 formation and 349 for C2H2 reduction. Activity of the Fe protein was O2-labile with a t1/2 of 36 s in air. At low temperatures the dithionite-reduced protein exhibited e.p.r. signals consistent with the presence of both S = 1/2 and S = 3/2 spin states. These signals were similar to those given by other nitrogenase Fe proteins, as were the changes in their line shape that occurred in the presence of MgATP or MgADP. The absorbance spectra showed that an increase in absorption occurred in the visible range on reversible oxidation of the dithionite-reduced protein. The oxidized-minus-reduced epsilon 420 was 6000 M-1.cm-1.
Eady
R R
RR
A.F.R.C.-Institute of Plant Science Research Nitrogen Fixation Laboratory, University of Sussex, Brighton, U.K.
Richardson
T H
TH
Miller
R W
RW
Hawkins
M
M
Lowe
D J
DJ
D000596:Amino Acids / Q000032:analysis; D001395:Azotobacter / Q000201:enzymology*; D002852:Chromatography, Ion Exchange; D004578:Electron Spin Resonance Spectroscopy; D004591:Electrophoresis, Polyacrylamide Gel; D007501:Iron / Q000032:analysis; D008667:Metalloproteins / Q000032:analysis*; D008970:Molecular Weight; D009591:Nitrogenase / Q000302:isolation & purification* / Q000378:metabolism; D010084:Oxidation-Reduction; D010446:Peptide Fragments / Q000032:analysis; D013053:Spectrophotometry; D013440:Sulfides / Q000032:analysis
D000596:Amino Acids; D008667:Metalloproteins; D010446:Peptide Fragments; D013440:Sulfides; D007501:Iron; D009591:Nitrogenase