Abstract
A major hurdle in the studies of nitrogenase, one of the most complicated metalloenzymes known to date, is to obtain large amounts of intact, active proteins. Nitrogenase and related proteins are often multimeric and consist of metal centers that are critical for their activities. Most notably, the well-studied MoFe protein of Mo-nitrogenase is a heterotetramer that houses two of the most complicated metal clusters found in nature, the P-cluster and the FeMoco (or M-cluster). The structural complexity of these proteins and the oxygen sensitivity of their associated metal clusters, along with the demand for large amounts of high-quality proteins in most downstream analyses, make large-scale, high-yield purification of fully competent nitrogenase proteins a formidable task and yet, at the same time, a prerequisite for the success of nitrogenase research. This chapter highlights several methods that have been developed over the past few decades chiefly for the purification of naturally expressed nitrogenase in the diazotroph Azotobacter vinelandii. In addition, purification and Fe-S reconstitution strategies are also outlined for the heterologously expressed nitrogenase proteins in Escherichia coli.
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References
Burgess BK, Wherland S, Stiefel EI et al (1980) HD formation by nitrogenase: a probe for N2 reduction intermediates. In: Newton WE, Otsuka S (eds) Molybdenum chemistry of biological significance. Plenum Press, New York
Stiefel EI, Burgess BK, Wherland S et al (1980) Azotobacter vinelandii biochemistry: H2(D2) relationships and some aspects of iron metabolism. In: Newton WE, Orme-Johnson WH (eds) Nitrogen fixation. University Park Press, Baltimore
Peters JW, Stowell MH, Soltis SM et al (1997) Redox-dependent structural changes in the nitrogenase P-cluster. Biochemistry 36:1181–1187
Georgiadis MM, Komiya H, Chakrabarti P et al (1992) Crystallographic structure of the nitrogenase iron protein from Azotobacter vinelandii. Science 257:1653–1659
Rawlings J, Shah VK, Chisnell JR et al (1978) Novel metal cluster in the iron-molybdenum cofactor of nitrogenase. Spectroscopic evidence. J Biol Chem 253:1001–1004
Cramer SP, Gillum WO, Hodgson KO et al (1978) The molybdenum site of nitrogenase. 2. A comparative study of molybdenum-iron proteins and the iron-molybdenum cofactor by x-ray absorption spectroscopy. J Am Chem Soc 100:3814–3819
Eady RR, Lowe DJ, Thorneley RNF (1978) Nitrogenase of Klebsiella pneumoniae: a pre-steady state burst of ATP hydrolysis is coupled to electron transfer between the component proteins. FEBS Lett 95:211–213
Burgess BK, Jacobs DB, Stiefel EI (1980) Large-scale purification of high activity Azotobacter vinelandii nitrogenase. Biochim Biophys Acta 614:196–209
Davis LC, Shah VK, Brill WJ (1975) Nitrogenase: VII. Effect of component ratio, ATP and H2, on the distribution of electrons to alternative substrates. Biochim Biophys Acta 403:67–78
Hu Y, Fay AW, Ribbe MW (2005) Identification of a nitrogenase FeMo cofactor precursor on NifEN complex. Proc Natl Acad Sci U S A 102:3236–3241
Wiig JA, Hu Y, Ribbe MW (2011) NifEN-B complex of Azotobacter vinelandii is fully functional in nitrogenase FeMo cofactor assembly. Proc Natl Acad Sci U S A 108:8623–8627
Lee CC, Hu Y, Ribbe MW (2009) Unique features of the nitrogenase VFe protein from Azotobacter vinelandii. Proc Natl Acad Sci U S A 23:9209–9214
Fay AW, Wiig JA, Lee CC, Hu Y (2015) Identification and characterization of functional homologs of nitrogenase cofactor biosynthesis protein NifB from methanogens. Proc Natl Acad Sci U S A 112:14829–14833
Acknowledgments
The authors are supported by the US Department of Energy (Basic Energy Sciences) grant DE-SC0016510 (to Y.H. and M.W.R.).
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Lee, CC., Ribbe, M.W., Hu, Y. (2019). Purification of Nitrogenase Proteins. In: Hu, Y. (eds) Metalloproteins. Methods in Molecular Biology, vol 1876. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-8864-8_7
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DOI: https://doi.org/10.1007/978-1-4939-8864-8_7
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