The vanadium nitrogenase of Azotobacter chroococcum. Purification and properties of the VFe protein

Abstract

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.

A Vanadium and Iron Cluster Accumulates on VnfX during Iron-Vanadium-cofactor Synthesis for the Vanadium Nitrogenase inAzotobacter vinelandii

“…Because the observation of this hyperfine structure is maximized at the same conditions as is the intensity of the S ϭ 3/2 species, it is probable that the hyperfine structure is due to 51 V incorporated into the species that produces the S ϭ 3/2 resonances, rather than due to a protein-bound 51 V 4ϩ (S ϭ 1/2) species. The ground state spin and spectral line shapes of the majority species resemble those of isolated FeV-co (28), the reduced vnf-dinitrogenase of A. chroococcum (29), and a synthetic [VFe 3 S 4 ] cubane cluster (30). The minority S ϭ 1/2 component (g ϭ 2.05, 1.96, 1.89) was most clearly observable at higher temperatures ( Fig.…”

Tuning a Nitrate Reductase for Function

“…The iron content was also determined in colorimetric assays using bipyridyl (28), and acid-labile sulfide was determined as described by Rasmussen et al (29).…”

Nucleotide sequence and mutational analysis of the structural genes (anfHDGK) for the second alternative nitrogenase from Azotobacter vinelandii

“…In A. vinelandii, nifHDK are organized in an operon that might contain four additional genes located downstream from nifK (57). Nitrogenase 2 from A. vinelandii and A. chroococcum is present in cells grown in N-free medium containing V (5,18,23,24,51,53). This nitrogenase also consists of two components.…”

“…This nitrogenase also consists of two components. Dinitrogenase reductase 2 is a dimer (18,23) whose subunits are thought to be encoded by vnfH (previously designated nifH2 [30] and nifH* [55]). Dinitrogenase 2 has been purified from A. vinelandii (24) and A. chroococcum (18) 14,000 appears to be present in dinitrogenase 2 from A. chroococcum (17,18).…”

“…Dinitrogenase reductase 2 is a dimer (18,23) whose subunits are thought to be encoded by vnfH (previously designated nifH2 [30] and nifH* [55]). Dinitrogenase 2 has been purified from A. vinelandii (24) and A. chroococcum (18) 14,000 appears to be present in dinitrogenase 2 from A. chroococcum (17,18). Dinitrogenase 2 contains Fe and V (18,24), and a FeV cofactor analogous to the FeMo cofactor has been reported for A. chroococcum (1,60).…”