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Production and isolation of vanadium nitrogenase from Azotobacter vinelandii by molybdenum depletion

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Abstract

The alternative, vanadium-dependent nitrogenase is employed by Azotobacter vinelandii for the fixation of atmospheric N2 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 ΔnifHDK 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 VnfD2K2G2 heterohexamer.

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Abbreviations

EPR:

Electron paramagnetic resonance

RALS:

Right-angle light scattering

SEC:

Size-exclusion chromatography

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Acknowledgements

The authors thank Eric Springer and Elke Gerbig-Smentek for assistance with cell growth and protein isolation. This work was supported by the European Research Council (ERC Grant No. 310656), the Deutsche Forschungsgemeinschaft (RTG 1976 and PP 1927) and the BIOSS Centre for Biological Signalling Studies.

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Authors and Affiliations

  1. Institute for Biochemistry, Albert-Ludwigs-University Freiburg, Albertstraße 21, 79104, Freiburg Im Breisgau, Germany

    Daniel Sippel, Julia Schlesier, Michael Rohde, Christian Trncik, Laure Decamps, Ivana Djurdjevic, Thomas Spatzal, Susana L. A. Andrade & Oliver Einsle

  2. BIOSS Centre for Biological Signalling Studies, 79104, Freiburg, Germany

    Daniel Sippel, Julia Schlesier, Michael Rohde, Christian Trncik, Laure Decamps, Ivana Djurdjevic, Thomas Spatzal, Susana L. A. Andrade & Oliver Einsle

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  1. Daniel Sippel
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Correspondence to Oliver Einsle.

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Sippel, D., Schlesier, J., Rohde, M. et al. Production and isolation of vanadium nitrogenase from Azotobacter vinelandii by molybdenum depletion. J Biol Inorg Chem 22, 161–168 (2017). https://doi.org/10.1007/s00775-016-1423-2

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  • DOI: https://doi.org/10.1007/s00775-016-1423-2

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