nitrogenase and tetrathiomolybdate

Molybdate- and ATP-dependent in vitro synthesis of the iron-molybdenum cofactor (FeMo-co) of nitrogenase requires the protein products of at least the nifB, nifN, and nifE genes. Extracts of FeMo-co-negative mutants of Klebsiella pneumoniae and Azotobacter vinelandii with lesions in different genes can be complemented for FeMo-co synthesis. Both K. pneumoniae and A. vinelandii dinitrogenase (component I) deficient in FeMo-co can be activated by FeMo-co synthesized in vitro. Properties of the partially purified dinitrogenase activated by FeMo-co synthesized in vitro were comparable to those of dinitrogenase from the wild-type organism; e.g., ratios of acetylene- to nitrogen-reduction activities, as well as those of acetylene reduction activities to EPR spectrum peak height at g = 3.65, were very similar. A. vinelandii mutants UW45 and CA30 have mutations in a gene functionally equivalent to nifB of K. pneumoniae.

Topics: Acetylene; Adenosine Triphosphate; Azotobacter; Bacterial Proteins; Cell-Free System; Chloramphenicol; Electron Spin Resonance Spectroscopy; Enzyme Activation; Ferredoxins; Genes, Bacterial; Genetic Complementation Test; Klebsiella pneumoniae; Molybdenum; Molybdoferredoxin; Nitrogen Fixation; Nitrogenase; Operon; Protein Processing, Post-Translational; Tungsten; Tungsten Compounds; Vanadates; Vanadium

Aerial oxidation of the iron-molybdenum cofactor (FeMoco) of Azotobacter vinelandii nitrogenase has been shown to yield either the tetrathiomolybdate ion ([MoS4]2-) or the oxotrithiomolybdate ion ([MoOS3]2-), depending on the reaction conditions. Thus, when N-methylformamide (NMF) solutions of FeMoco either were titrated with measured aliquots of air or were diluted with air-saturated NMF, [MoOS3]2- was found to be the predominant product while dilution of NMF solutions of FeMoco with air-saturated methanol produced [MoS4]2- almost exclusively. Similar aerial oxidation of solutions of chemically synthesized Fe-Mo-S clusters showed that significant information about the molybdenum environment in these species could be deduced from the nature of the elicited thiomolybdates. The differences in decomposition products as a function of solvent are postulated to be due to the loss through precipitation of the reducing agent sodium dithionite on addition of methanol but not NMF. These overall decomposition results are discussed in the context of recent X-ray absorption spectroscopic data which suggest the presence of an 'MoS3' core in FeMoco. A possible mechanism whereby [MoS4]2- might be rapidly formed from this core is presented.

Topics: Azotobacter; Coenzymes; Metalloproteins; Molybdenum; Molybdenum Cofactors; Nitrogenase; Oxidation-Reduction; Oxidoreductases; Pteridines; Spectrophotometry; Sulfur

Tetrathiomolybdate inhibits iron-molybdenum cofactor (FeMo cofactor) binding to component I of nitrogenase. Molybdenum-iron cluster (a subcomponent of FeMo cofactor) and tetrathiomolybdate inhibited FeMo cofactor activation of inactive nitrogenase component I in extracts of Azotobacter vinelandii and Klebsiella pneumoniae mutant strains defective in the biosynthesis of FeMo cofactor. Addition of tetrathiotungstate, the tungsten analog of tetrathiomolybdate, to the mutant extracts had no significant inhibitory effect on subsequent activation by FeMo cofactor.

Topics: Azotobacter; Enzyme Activation; Ferredoxins; Macromolecular Substances; Molybdenum; Molybdoferredoxin; Nitrogenase

The inactive MoFe protein (NifB-Kp1) of nitrogenase from nifB mutants of Klebsiella pneumoniae may be activated by addition of the iron-molybdenum cofactor (FeMoco) extracted from active MoFe protein (Kp1). However, when apparently saturated with FeMoco, our preparations of NifB-Kp1 yielded activated protein, Kp1-asm, with a specific activity that was at best only 40% of that expected. This was not due to degradation of Kp1-asm, NifB-Kp1 or FeMoco during the activation reaction. Nor could activation be enhanced by addition of other nif-gene products or other proteins. Whereas fully active Kp1 contains 2 FeMoco/molecule, apparent saturation of our NifB-Kp1 preparations required the binding of only 0.4-0.65 FeMoco/molecule. By using chromatography Kp1-asm could be largely resolved from NifB-Kp1 that had not been activated. However, we were unable to isolate fully active MoFe protein (i.e. Kp1-asm containing 2 FeMoco/molecule) from solutions of NifB-Kp1 activated with FeMoco. The maximum activity/ng-atom of total Mo obtained for our purified Kp1-asm was approximately half the maximum activity for FeMoco. Since all NifB-Kp1 preparations contained some Mo, we suggest that FeMoco activated only those NifB-Kp1 molecules already containing one atom of (non-FeMoco) Mo, thus forming Kp1-asm with 2 Mo but only 1 FeMoco/molecule. Kp1-asm was identical with normal Kp1 in terms of its Mr, stability, e.p.r. signals, pattern of substrate reductions, CO inhibition and ATP/2e ratio. In addition, for preparations of differing specific activity, there was a constant and identical relationship between the e.p.r. signal intensity (from FeMoco) and the activity of both Kp1 and Kp1-asm. Assuming the above hypothesis on the structure of Kp1-asm, these data demonstrate that the two FeMoco sites in wild-type Kp1 operate independently.

Topics: Chromatography, DEAE-Cellulose; Electron Spin Resonance Spectroscopy; Enzyme Activation; Ferredoxins; Klebsiella pneumoniae; Molybdenum; Molybdoferredoxin; Mutation; Nitrogenase

Topics: Clostridium; Molybdenum; Nitrogenase; Spectrum Analysis, Raman