The vanadium nitrogenase of Azotobacter

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The properties of the recently characterized vanadium-dependent nitrogenase system of Azotobacter are reviewed and compared with the better characterized Mo-nitrogenases. Vanadium is present in V-nitrogenase in a vanadium- and iron-containing protein (VFe protein), analogous in structure and function to the MoFe protein of Mo-nitrogenase. The VFe protein contains vanadium in a polynuclear cluster in an environment with iron, sulphur and oxygen (or nitrogen or carbon) as nearest neighbour atoms, very similar to the environment of molybdenum in the iron and molybdenum cofactor (FeMoco) centre of the MoFe proteins. A vanadium-containing cluster can be extracted from the VFe protein by treatment with N-methylformamide (NMF). The NMF extract contains vanadium, iron and sulphur in a 1:6:5 ratio and activates the inactive MoFe protein synthesized by some mutant organisms unable to synthesize FeMoco. This hybrid protein has the characteristic substrate reduction pattern of a VFe protein, in forming some C 2 H 6 from C 2 H 2 . These data are consistent with the NMF extract containing an iron and vanadium cofactor FeVaco, analogous to FeMoco. Although the hybrid protein is active in nitrogenase assays with H + or C 2 H 2 as reducible substrates, N 2 is not reduced, suggesting that specific interactions of the cofactor centre with a polypeptide ligand are lacking. Pre-steady-state kinetics of the ATP-dependent electron-transfer from the Fe protein of V-nitrogenase to a VFe protein are very similar to those of Mo-nitrogenase components, consistent with a common mechanism of substrate reduction. However, at low temperatures N 2 is a more effective substrate for V-nitrogenase compared with Mo-nitrogenase.