Journal
JOURNAL OF BIOLOGICAL INORGANIC CHEMISTRY
Volume 22, Issue 1, Pages 161-168Publisher
SPRINGER
DOI: 10.1007/s00775-016-1423-2
Keywords
Biological nitrogen fixation; Alternative nitrogenase; Vanadium; VFe protein; Anoxic protein biochemistry
Funding
- European Research Council (ERC) [310656]
- Deutsche Forschungsgemeinschaft [RTG 1976, PP 1927]
- BIOSS Centre for Biological Signalling Studies
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The alternative, vanadium-dependent nitrogenase is employed by Azotobacter vinelandii for the fixation of atmospheric N-2 under conditions of molybdenum starvation. While overall similar in architecture and functionality to the common Mo-nitrogenase, the V-dependent enzyme exhibits a series of unique features that on one hand are of high interest for biotechnological applications. As its catalytic properties differ from Mo-nitrogenase, it may on the other hand also provide invaluable clues regarding the molecular mechanism of biological nitrogen fixation that remains scarcely understood to date. Earlier studies on vanadium nitrogenase were almost exclusively based on a Delta nifHDK strain of A. vinelandii, later also in a version with a hexahistidine affinity tag on the enzyme. As structural analyses remained unsuccessful with such preparations we have developed protocols to isolate unmodified vanadium nitrogenase from molybdenum-depleted, actively nitrogen-fixing A. vinelandii wild-type cells. The procedure provides pure protein at high yields whose spectroscopic properties strongly resemble data presented earlier. Analytical size-exclusion chromatography shows this preparation to be a VnfD(2)K(2)G(2) heterohexamer.
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