The vanadium nitrogenase of Azotobacter chroococcum. Purification and properties of the VFe protein.
1. Nitrogenase activity of a strain of Azotobacter chroococcum lacking the structural genes for conventional nitrogenase (nifHDK) was separated into two components: an Fe-containing protein and a vanadoprotein. 2. The larger protein was purified to homogeneity by the criterion of electrophoresis of 10% (w/v) acrylamide gels in the presence of SDS. Two types of subunit, of Mr 50,000 and 55,000, were present in equal amounts. 3. The protein had an Mr of 210,000 and contained 2 V atoms, 23 Fe atoms and 20 acid-labile sulphide groups per molecule. The Mo content was less than 0.06 g-atom/mol. All the common amino acids were present, with a predominance of acidic residues. Ultracentrifugal analysis gave a maximum sedimentation coefficient of 9.7 S and a symmetrical boundary at 5 mg of protein X ml-1; dissociation occurred at lower concentrations. The specific activities (nmol of product/min per mg of protein), when assayed under optimum conditions with the complementary Fe protein from this strain, were 1348 for H2 evolution, 350 for NH3 formation and 608 for acetylene reduction. Activity was O2-labile, with a t1/2 of 40 s in air. At low temperatures the dithionite-reduced protein showed e.p.r. signals at g = 5.6, 4.35, 3.77 and 1.93, consistent with an S = 3/2 ground state with an additional S = 1/2 centre giving rise to the feature at g = 1.93. The u.v. spectra of dithionite-reduced and thionine-oxidized protein were very similar. Oxidation resulted in a general increase in absorbance in the visible region. The shoulder at 380 nm in the spectrum of reduced protein was replaced with shoulders near 330 nm and 420 nm on oxidation.
Eady
R R
RR
AFRC Unit of Nitrogen Fixation, University of Sussex, Brighton, U.K.
Robson
R L
RL
Richardson
T H
TH
Miller
R W
RW
Hawkins
M
M
D000596:Amino Acids / Q000032:analysis; D001395:Azotobacter / Q000201:enzymology*; D002499:Centrifugation, Density Gradient; D002850:Chromatography, Gel; D004578:Electron Spin Resonance Spectroscopy; D004591:Electrophoresis, Polyacrylamide Gel; D009591:Nitrogenase / Q000302:isolation & purification* / Q000378:metabolism; D010446:Peptide Fragments / Q000032:analysis; D013053:Spectrophotometry; D013379:Substrate Specificity; D014639:Vanadium / Q000032:analysis
D000596:Amino Acids; D010446:Peptide Fragments; D014639:Vanadium; D009591:Nitrogenase