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Fe-S Proteins pp 97–121Cite as

Expression, Isolation, and Characterization of Vanadium Nitrogenase from Azotobacter vinelandii

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Part of the book series: Methods in Molecular Biology ((MIMB,volume 2353))

Abstract

Nitrogenases are the sole enzymes known to mediate biological nitrogen fixation, an essential process for sustaining life on earth. Among the three known variants, molybdenum nitrogenase is the best-studied to date. Recent work on the alternative vanadium nitrogenase provided important insights into the mechanism of nitrogen fixation since this enzyme differs from its molybdenum counterpart in some important aspects. Here, we present a protocol to obtain unmodified vanadium nitrogenase in high yield and purity from the paradigmatic diazotroph Azotobacter vinelandii, including procedures for cell cultivation, purification, and protein characterization.

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References

  1. Howard JB, Rees DC (1996) Structural basis of biological nitrogen fixation. Chem Rev 96(7):2965–2982. https://doi.org/10.1021/cr9500545

    Article  CAS  PubMed  Google Scholar 

  2. Tamaru K (1991) The history of the development of ammonia synthesis. In: Catalytic ammonia synthesis. Springer, Berlin, pp 1–18

    Google Scholar 

  3. Erisman JW, Sutton MA, Galloway J, Klimont Z, Winiwarter W (2008) How a century of ammonia synthesis changed the world. Nat Geosci 1(10):636–639. https://doi.org/10.1038/ngeo325

    Article  CAS  Google Scholar 

  4. Schrock RR (2006) Reduction of dinitrogen. Proc Natl Acad Sci U S A 103(46):17087. https://doi.org/10.1073/pnas.0603633103

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Lipman JG (1903) Experiments on the transformation and fixation of nitrogen by bacteria. New Jersey State Agric Exp Sta Ann Rep 24:217–285

    Google Scholar 

  6. Rees DC (1993) Dinitrogen reduction by nitrogenase: if N2 isn't broken, it can't be fixed. Curr Opin Struct Biol 3(6):921–928. https://doi.org/10.1016/0959-440x(93)90157-g

    Article  CAS  Google Scholar 

  7. Dos Santos PC, Fang Z, Mason SW, Setubal JC, Dixon R (2012) Distribution of nitrogen fixation and nitrogenase-like sequences amongst microbial genomes. BMC Genomics 13(1):162. https://doi.org/10.1186/1471-2164-13-162

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Dos Santos PC, Addo MA (2020) Distribution of nitrogen fixation genes in prokaryotes containing alternative nitrogenases. Chembiochem 21(12):1749–1759. https://doi.org/10.1002/cbic.202000022

    Article  CAS  PubMed  Google Scholar 

  9. Bishop PE, Jarlenski DM, Hetherington DR (1980) Evidence for an alternative nitrogen fixation system in Azotobacter vinelandii. Proc Natl Acad Sci U S A 77(12):7342–7346. https://doi.org/10.1073/pnas.77.12.7342

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Chisnell JR, Premakumar R, Bishop PE (1988) Purification of a second alternative nitrogenase from a nifHDK deletion strain of Azotobacter vinelandii. J Bacteriol 170(1):27–33. https://doi.org/10.1128/jb.170.1.27-33.1988

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Bulen WA, LeComte JR (1966) The nitrogenase system from Azotobacter: two-enzyme requirement for N2 reduction, ATP-dependent H2 evolution, and ATP hydrolysis. Proc Natl Acad Sci 56(3):979–986. https://doi.org/10.1073/pnas.56.3.979

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Brigle KE, Newton WE, Dean DR (1985) Complete nucleotide sequence of the Azotobacter vinelandii nitrogenase structural gene cluster. Gene 37(1–3):37–44. https://doi.org/10.1016/0378-1119(85)90255-0

    Article  CAS  PubMed  Google Scholar 

  13. Joerger RD, Loveless TM, Pau RN, Mitchenall LA, Simon BH, Bishop PE (1990) Nucleotide sequences and mutational analysis of the structural genes for nitrogenase 2 of Azotobacter vinelandii. J Bacteriol 172(6):3400–3408. https://doi.org/10.1128/jb.172.6.3400-3408.1990

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Joerger RD, Jacobson MR, Premakumar R, Wolfinger ED, Bishop PE (1989) Nucleotide sequence and mutational analysis of the structural genes (anfHDGK) for the second alternative nitrogenase from Azotobacter vinelandii. J Bacteriol 171(2):1075–1086. https://doi.org/10.1128/jb.171.2.1075-1086.1989

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Boyd E, Hamilton T, Peters J (2011) An alternative path for the evolution of biological nitrogen fixation. Front Microbiol 2:205. https://doi.org/10.3389/fmicb.2011.00205

    Article  PubMed  PubMed Central  Google Scholar 

  16. Stueken EE, Buick R, Guy BM, Koehler MC (2015) Isotopic evidence for biological nitrogen fixation by molybdenum-nitrogenase from 3.2 Gyr. Nature 520(7549):666–669. https://doi.org/10.1038/nature14180

    Article  CAS  PubMed  Google Scholar 

  17. Anbar AD, Knoll AH (2002) Proterozoic Ocean chemistry and evolution: a bioinorganic bridge? Science 297(5584):1137–1142. https://doi.org/10.1126/science.1069651

    Article  CAS  PubMed  Google Scholar 

  18. Hans Wedepohl K (1995) The composition of the continental crust. Geochim Cosmochim Acta 59(7):1217–1232. https://doi.org/10.1016/0016-7037(95)00038-2

    Article  Google Scholar 

  19. Bellenger JP, Wichard T, Kraepiel AM (2008) Vanadium requirements and uptake kinetics in the dinitrogen-fixing bacterium Azotobacter vinelandii. Appl Environ Microbiol 74(5):1478–1484. https://doi.org/10.1128/AEM.02236-07

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Boyd ES, Anbar AD, Miller S, Hamilton TL, Lavin M, Peters JW (2011) A late methanogen origin for molybdenum-dependent nitrogenase. Geobiology 9(3):221–232. https://doi.org/10.1111/j.1472-4669.2011.00278.x

    Article  CAS  PubMed  Google Scholar 

  21. Raymond J, Siefert JL, Staples CR, Blankenship RE (2004) The natural history of nitrogen fixation. Mol Biol Evol 21(3):541–554. https://doi.org/10.1093/molbev/msh047

    Article  CAS  PubMed  Google Scholar 

  22. Noar JD, Bruno-Bárcena JM (2018) Azotobacter vinelandii: the source of 100 years of discoveries and many more to come. Microbiology 164(4):421–436. https://doi.org/10.1099/mic.0.000643

    Article  CAS  PubMed  Google Scholar 

  23. Pau RN, Mitchenall LA, Robson RL (1989) Genetic evidence for an Azotobacter vinelandii nitrogenase lacking molybdenum and vanadium. J Bacteriol 171(1):124–129. https://doi.org/10.1128/jb.171.1.124-129.1989

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Eady RR (1996) Structureminus sign function relationships of alternative Nitrogenases. Chem Rev 96(7):3013–3030. https://doi.org/10.1021/cr950057h

    Article  CAS  PubMed  Google Scholar 

  25. Setubal JC, dos Santos P, Goldman BS, Ertesvag H, Espin G, Rubio LM, Valla S, Almeida NF, Balasubramanian D, Cromes L, Curatti L, Du Z, Godsy E, Goodner B, Hellner-Burris K, Hernandez JA, Houmiel K, Imperial J, Kennedy C, Larson TJ, Latreille P, Ligon LS, Lu J, Maerk M, Miller NM, Norton S, O'Carroll IP, Paulsen I, Raulfs EC, Roemer R, Rosser J, Segura D, Slater S, Stricklin SL, Studholme DJ, Sun J, Viana CJ, Wallin E, Wang B, Wheeler C, Zhu H, Dean DR, Dixon R, Wood D (2009) Genome sequence of Azotobacter vinelandii, an obligate aerobe specialized to support diverse anaerobic metabolic processes. J Bacteriol 191(14):4534–4545. https://doi.org/10.1128/JB.00504-09

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Rediers H, Vanderleyden J, De Mot R (2004) Azotobacter vinelandii: a pseudomonas in disguise? Microbiology 150(Pt 5):1117–1119. https://doi.org/10.1099/mic.0.27096-0

    Article  CAS  PubMed  Google Scholar 

  27. Setubal JC, Almeida NF (2015) The Azotobacter vinelandii genome: an update. In: Biological nitrogen fixation, vol 1 and 2. John Wiley & Sons, Inc., Hoboken, New Jersey, pp 225–234. https://doi.org/10.1002/9781119053095

    Chapter  Google Scholar 

  28. Young JM, Park D-C (2007) Probable synonymy of the nitrogen-fixing genus Azotobacter and the genus pseudomonas. Int J Syst Evol Microbiol 57(12):2894–2901. https://doi.org/10.1099/ijs.0.64969-0

    Article  CAS  PubMed  Google Scholar 

  29. Özen AI, Ussery DW (2012) Defining the pseudomonas genus: where do we draw the line with Azotobacter? Microb Ecol 63(2):239–248. https://doi.org/10.1007/s00248-011-9914-8

    Article  PubMed  Google Scholar 

  30. Robson RL, Eady RR, Richardson TH, Miller RW, Hawkins M, Postgate JR (1986) The alternative nitrogenase of Azotobacter chroococcum is a vanadium enzyme. Nature 322(6077):388–390. https://doi.org/10.1038/322388a0

    Article  CAS  Google Scholar 

  31. McKenna CE, Benemann JR, Traylor TG (1970) A vanadium containing nitrogenase preparation: implications for the role of molybdenum in nitrogen fixation. Biochem Biophys Res Commun 41(6):1501–1508. https://doi.org/10.1016/0006-291x(70)90557-7

    Article  CAS  PubMed  Google Scholar 

  32. Bishop PE, Jarlenski DM, Hetherington DR (1982) Expression of an alternative nitrogen fixation system in Azotobacter vinelandii. J Bacteriol 150(3):1244–1251

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Burns RC, Fuchsman WH, Hardy RWF (1971) Nitrogenase from vanadium-grown : isolation, characteristics, and mechanistic implications. Biochem Biophys Res Commun 42(3):353–358. https://doi.org/10.1016/0006-291x(71)90377-9

    Article  CAS  PubMed  Google Scholar 

  34. Premakumar R, Lemos E, Bishop P (1984) Evidence for two dinitrogenase reductases under regulatory control by molybdenum in Azotobacter vinelandii☆. Biochim Biophys Acta Gen Subj 797(1):64–70. https://doi.org/10.1016/0304-4165(84)90382-9

    Article  CAS  Google Scholar 

  35. Bishop PE, Hawkins ME, Eady RR (1986) Nitrogen fixation in molybdenum-deficient continuous culture by a strain of Azotobacter vinelandii carrying a deletion of the structural genes for nitrogenase (nifHDK). Biochem J 238(2):437–442. https://doi.org/10.1042/bj2380437

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Joerger RD, Premakumar R, Bishop PE (1986) Tn5-induced mutants of Azotobacter vinelandii affected in nitrogen fixation under Mo-deficient and Mo-sufficient conditions. J Bacteriol 168(2):673–682. https://doi.org/10.1128/jb.168.2.673-682.1986

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Bishop PE, Premakumar R, Dean DR, Jacobson MR, Chisnell JR, Rizzo TM, Kopczynski J (1986) Nitrogen fixation by Azotobacter vinelandii strains having deletions in structural genes for Nitrogenase. Science 232(4746):92–94. https://doi.org/10.1126/science.232.4746.92

    Article  CAS  PubMed  Google Scholar 

  38. Jacobson MR, Premakumar R, Bishop PE (1986) Transcriptional regulation of nitrogen fixation by molybdenum in Azotobacter vinelandii. J Bacteriol 167(2):480–486. https://doi.org/10.1128/jb.167.2.480-486.1986

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Luque F, Pau RN (1991) Transcriptional regulation by metals of structural genes for Azotobacter vinelandii nitrogenases. Mol Gen Genet MGG 227(3):481–487. https://doi.org/10.1007/BF00273941

    Article  CAS  PubMed  Google Scholar 

  40. Waugh SI, Paulsen DM, Mylona PV, Maynard RH, Premakumar R, Bishop PE (1995) The genes encoding the delta subunits of dinitrogenases 2 and 3 are required for mo-independent diazotrophic growth by Azotobacter vinelandii. J Bacteriol 177(6):1505–1510. https://doi.org/10.1128/jb.177.6.1505-1510.1995

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Walmsley J, Kennedy C (1991) Temperature-dependent regulation by molybdenum and vanadium of expression of the structural genes encoding three Nitrogenases in Azotobacter vinelandii. Appl Environ Microbiol 57(2):622–624

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Miller RW, Eady RR (1988) Molybdenum and vanadium nitrogenases of Azotobacter chroococcum. Low temperature favours N2 reduction by vanadium nitrogenase. Biochem J 256(2):429–432. https://doi.org/10.1042/bj2560429

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Luxem KE, Kraepiel AML, Zhang L, Waldbauer JR, Zhang X (2020) Carbon substrate re-orders relative growth of a bacterium using Mo-, V-, or Fe-nitrogenase for nitrogen fixation. Environ Microbiol 22(4):1397–1408. https://doi.org/10.1111/1462-2920.14955

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Darnajoux R, Magain N, Renaudin M, Lutzoni F, Bellenger JP, Zhang X (2019) Molybdenum threshold for ecosystem scale alternative vanadium nitrogenase activity in boreal forests. Proc Natl Acad Sci U S A 116(49):24682–24688. https://doi.org/10.1073/pnas.1913314116

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Kim JS, Rees DC (1992) Crystallographic structure and functional implications of the Nitrogenase molybdenum iron protein from Azotobacter-Vinelandii. Nature 360(6404):553–560

    Article  CAS  PubMed  Google Scholar 

  46. Georgiadis MM, Komiya H, Chakrabarti P, Woo D, Kornuc JJ, Rees DC (1992) Crystallographic structure of the nitrogenase iron protein from Azotobacter vinelandii. Science 257(5077):1653–1659. https://doi.org/10.1126/science.1529353

    Article  CAS  PubMed  Google Scholar 

  47. Schindelin H, Kisker C, Schlessman JL, Howard JB, Rees DC (1997) Structure of ADP·AIF4–stabilized nitrogenase complex and its implications for signal transduction. Nature 387(6631):370–376. https://doi.org/10.1038/387370a0

    Article  CAS  PubMed  Google Scholar 

  48. Einsle O, Tezcan FA, Andrade SL, Schmid B, Yoshida M, Howard JB, Rees DC (2002) Nitrogenase MoFe-protein at 1.16 a resolution: a central ligand in the FeMo-cofactor. Science 297(5587):1696–1700. https://doi.org/10.1126/science.1073877

    Article  CAS  PubMed  Google Scholar 

  49. Spatzal T, Aksoyoglu M, Zhang L, Andrade SL, Schleicher E, Weber S, Rees DC, Einsle O (2011) Evidence for interstitial carbon in nitrogenase FeMo cofactor. Science 334(6058):940. https://doi.org/10.1126/science.1214025

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. Lancaster KM, Roemelt M, Ettenhuber P, Hu Y, Ribbe MW, Neese F, Bergmann U, DeBeer S (2011) X-ray emission spectroscopy evidences a central carbon in the nitrogenase iron-molybdenum cofactor. Science 334(6058):974–977. https://doi.org/10.1126/science.1206445

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  51. Rees JA, Bjornsson R, Schlesier J, Sippel D, Einsle O, DeBeer S (2015) The Fe-V cofactor of vanadium Nitrogenase contains an interstitial carbon atom. Angew Chem Int Ed Engl 54(45):13249–13252. https://doi.org/10.1002/anie.201505930

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  52. Sippel D, Einsle O (2017) The structure of vanadium nitrogenase reveals an unusual bridging ligand. Nat Chem Biol 13(9):956–960. https://doi.org/10.1038/nchembio.2428

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  53. Rohde M, Trncik C, Sippel D, Gerhardt S, Einsle O (2018) Crystal structure of VnfH, the iron protein component of vanadium nitrogenase. J Biol Inorg Chem 23(7):1049–1056. https://doi.org/10.1007/s00775-018-1602-4

    Article  CAS  PubMed  Google Scholar 

  54. Sippel D, Schlesier J, Rohde M, Trncik C, Decamps L, Djurdjevic I, Spatzal T, Andrade SL, Einsle O (2017) Production and isolation of vanadium nitrogenase from Azotobacter vinelandii by molybdenum depletion. J Biol Inorg Chem 22(1):161–168. https://doi.org/10.1007/s00775-016-1423-2

    Article  CAS  PubMed  Google Scholar 

  55. Peters JW, Stowell MH, Soltis SM, Finnegan MG, Johnson MK, Rees DC (1997) Redox-dependent structural changes in the nitrogenase P-cluster. Biochemistry 36(6):1181–1187. https://doi.org/10.1021/bi9626665

    Article  CAS  PubMed  Google Scholar 

  56. Eady R (2003) Current status of structure function relationships of vanadium nitrogenase. Coord Chem Rev 237(1–2):23–30. https://doi.org/10.1016/s0010-8545(02)00248-5

    Article  CAS  Google Scholar 

  57. Benediktsson B, Thorhallsson AT, Bjornsson R (2018) QM/MM calculations reveal a bridging hydroxo group in a vanadium nitrogenase crystal structure. Chem Commun (Camb) 54(53):7310–7313. https://doi.org/10.1039/c8cc03793k

    Article  CAS  Google Scholar 

  58. Hales BJ, Case EE, Morningstar JE, Dzeda MF, Mauterer LA (1986) Isolation of a new vanadium-containing nitrogenase from Azotobacter vinelandii. Biochemistry 25(23):7251–7255. https://doi.org/10.1021/bi00371a001

    Article  CAS  PubMed  Google Scholar 

  59. Chatterjee R, Allen RM, Ludden PW, Shah VK (1997) In vitro synthesis of the iron-molybdenum cofactor and maturation of the nif-encoded apodinitrogenase. Effect of substitution of VNFH for NIFH. J Biol Chem 272(34):21604–21608. https://doi.org/10.1074/jbc.272.34.21604

    Article  CAS  PubMed  Google Scholar 

  60. Walker JE, Saraste M, Runswick MJ, Gay NJ (1982) Distantly related sequences in the alpha- and beta-subunits of ATP synthase, myosin, kinases and other ATP-requiring enzymes and a common nucleotide binding fold. EMBO J 1(8):945–951. https://doi.org/10.1002/j.1460-2075.1982.tb01276.x

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  61. Danyal K, Dean DR, Hoffman BM, Seefeldt LC (2011) Electron transfer within nitrogenase: evidence for a deficit-spending mechanism. Biochemistry 50(43):9255–9263. https://doi.org/10.1021/bi201003a

    Article  CAS  PubMed  Google Scholar 

  62. Peters JW, Fisher K, Dean DR (1995) Nitrogenase structure and function: a biochemical-genetic perspective. Annu Rev Microbiol 49(1):335–366. https://doi.org/10.1146/annurev.mi.49.100195.002003

    Article  CAS  PubMed  Google Scholar 

  63. Thorneley RNF, Lowe DJ (1983) Nitrogenase of Klebsiella pneumoniae. Kinetics of the dissociation of oxidized iron protein from molybdenum-iron protein: identification of the rate-limiting step for substrate reduction. Biochem J 215(2):393–403. https://doi.org/10.1042/bj2150393

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  64. Simpson F, Burris R (1984) A nitrogen pressure of 50 atmospheres does not prevent evolution of hydrogen by nitrogenase. Science 224(4653):1095–1097. https://doi.org/10.1126/science.6585956

    Article  CAS  PubMed  Google Scholar 

  65. Harris DF, Lukoyanov DA, Shaw S, Compton P, Tokmina-Lukaszewska M, Bothner B, Kelleher N, Dean DR, Hoffman BM, Seefeldt LC (2018) Mechanism of N2 reduction catalyzed by Fe-Nitrogenase involves reductive elimination of H2. Biochemistry 57(5):701–710. https://doi.org/10.1021/acs.biochem.7b01142

    Article  CAS  PubMed  Google Scholar 

  66. Harris DF, Lukoyanov DA, Kallas H, Trncik C, Yang ZY, Compton P, Kelleher N, Einsle O, Dean DR, Hoffman BM, Seefeldt LC (2019) Mo-, V-, and Fe-Nitrogenases use a universal eight-electron reductive-elimination mechanism to achieve N2 reduction. Biochemistry 58(30):3293–3301. https://doi.org/10.1021/acs.biochem.9b00468

    Article  CAS  PubMed  Google Scholar 

  67. Dilworth MJ (1966) Acetylene reduction by nitrogen-fixing preparations from clostridium pasteurianum. Biochim Biophys Acta Gen Subj 127(2):285–294. https://doi.org/10.1016/0304-4165(66)90383-7

    Article  CAS  Google Scholar 

  68. R S Burris R Study Intermediates in nitrogen fixation. In: Federation Proceedings, 1966. vol 2 P 1. Federation Amer Soc Exp Biol 9650, Rockville Pike, Bethesda, MD 20814–3998, p. 710

    Google Scholar 

  69. Harris DF, Yang ZY, Dean DR, Seefeldt LC, Hoffman BM (2018) Kinetic understanding of N2 reduction versus H2 evolution at the E4(4H) Janus state in the three Nitrogenases. Biochemistry 57(39):5706–5714. https://doi.org/10.1021/acs.biochem.8b00784

    Article  CAS  PubMed  Google Scholar 

  70. Riddle GD, Simonson JG, Hales BJ, Braymer HD (1982) Nitrogen fixation system of tungsten-resistant mutants of Azotobacter vinelandii. J Bacteriol 152(1):72–80

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  71. Dilworth MJ, Eady RR, Robson RL, Miller RW (1987) Ethane formation from acetylene as a potential test for vanadium nitrogenase in vivo. Nature 327(6118):167–168. https://doi.org/10.1038/327167a0

    Article  CAS  Google Scholar 

  72. Schollhorn R, Burris RH (1967) Acetylene as a competitive inhibitor of N-2 fixation. Proc Natl Acad Sci U S A 58(1):213–216. https://doi.org/10.1073/pnas.58.1.213

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  73. Dilworth MJ, Eady RR (1991) Hydrazine is a product of dinitrogen reduction by the vanadium-nitrogenase from Azotobacter chroococcum. Biochem J 277(Pt 2):465–468. https://doi.org/10.1042/bj2770465

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  74. Benemann J, McKenna C, Lie R, Traylor T, Kamen M (1972) The vanadium effect in nitrogen fixation by azotobacter. Biochim Biophys Acta Gen Subj 264(1):25–38. https://doi.org/10.1016/0304-4165(72)90113-4

    Article  CAS  Google Scholar 

  75. Dilworth MJ, Eady RR, Eldridge ME (1988) The vanadium nitrogenase of Azotobacter chroococcum. Reduction of acetylene and ethylene to ethane. Biochem J 249(3):745–751. https://doi.org/10.1042/bj2490745

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  76. Smith BE, Eady RR (1993) Metalloclusters of the nitrogenases. In: EJB reviews. Springer, Berlin, Heidelberg, pp 79–93. https://doi.org/10.1007/978-3-642-78046-2_7

    Chapter  Google Scholar 

  77. Scherings G, Haaker H, Veeger C (1977) Regulation of nitrogen fixation by Fe-S protein II in Azotobacter vinelandii. Eur J Biochem 77(3):21–30. https://doi.org/10.1111/j.1432-1033.1977.tb11706.x

    Article  CAS  PubMed  Google Scholar 

  78. Robson RL (1979) Characterization of an oxygen-stable nitrogenase complex isolated from Azotobacter chroococcum. Biochem J 181(3):569–575. https://doi.org/10.1042/bj1810569

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  79. Kuhla J, Oelze J (1988) Dependence of nitrogenase switch-off upon oxygen stress on the nitrogenase activity in Azotobacter vinelandii. J Bacteriol 170(11):5325–5329. https://doi.org/10.1128/jb.170.11.5325-5329.1988

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  80. Schlesier J, Rohde M, Gerhardt S, Einsle O (2016) A conformational switch triggers Nitrogenase protection from oxygen damage by Shethna protein II (FeSII). J Am Chem Soc 138(1):239–247. https://doi.org/10.1021/jacs.5b10341

    Article  CAS  PubMed  Google Scholar 

  81. Eady RR, Robson RL, Richardson TH, Miller RW, Hawkins M (1987) The vanadium nitrogenase of Azotobacter chroococcum. Purification and properties of the VFe protein. Biochem J 244(1):197–207. https://doi.org/10.1042/bj2440197

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  82. Burns RC, Stasny JT, Hardy RWF (1976). Isolation and characteristics of a modified nitrogenase from Azotobacter vinelandii including “vanadium-Fe” protein from cells grown on medium enriched in vanadium. Vol. 1 p.196–207. In W. E. Newton, C. J. Nyman (ed.), Proceedings of the First International Symposium on Nitrogen Fixation, Washington State University Press

    Google Scholar 

  83. Hales BJ, Langosch DJ, Case EE (1986) Isolation and characterization of a second nitrogenase Fe-protein from Azotobacter vinelandii. J Biol Chem 261(32):15301–15306

    Article  CAS  PubMed  Google Scholar 

  84. Blanchard CZ, Hales BJ (1996) Isolation of two forms of the nitrogenase VFe protein from Azotobacter vinelandii. Biochemistry 35(2):472–478. https://doi.org/10.1021/bi951429j

    Article  CAS  PubMed  Google Scholar 

  85. Robson RL, Woodley PR, Pau RN, Eady RR (1989) Structural genes for the vanadium nitrogenase from Azotobacter chroococcum. EMBO J 8(4):1217–1224. https://doi.org/10.1002/j.1460-2075.1989.tb03495.x

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  86. Chatterjee R, Allen RM, Ludden PW, Shah VK (1996) Purification and characterization of the vnf-encoded Apodinitrogenase from Azotobacter vinelandii. J Biol Chem 271(12):6819–6826. https://doi.org/10.1074/jbc.271.12.6819

    Article  CAS  PubMed  Google Scholar 

  87. Pau RN, Eldridge ME, Lowe DJ, Mitchenall LA, Eady RR (1993) Molybdenum-independent nitrogenases of Azotobacter vinelandii: a functional species of alternative nitrogenase-3 isolated from a molybdenum-tolerant strain contains an iron-molybdenum cofactor. Biochem J 293(Pt 1):101–107. https://doi.org/10.1042/bj2930101

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  88. Lee CC, Hu YL, Ribbe MW (2009) Unique features of the nitrogenase VFe protein from Azotobacter vinelandii. Proc Natl Acad Sci U S A 106(23):9209–9214. https://doi.org/10.1073/pnas.0904408106

    Article  PubMed  PubMed Central  Google Scholar 

  89. Chatterjee R, Ludden PW, Shah VK (1997) Characterization of VNFG, THE δ subunit of the vnf-encoded Apodinitrogenase from Azotobacter vinelandii: implications for its role in the formation of functional dinitrogenase 2. J Biol Chem 272(6):3758–3765. https://doi.org/10.1074/jbc.272.6.3758

    Article  CAS  PubMed  Google Scholar 

  90. Burk D, Lineweaver H (1930) The influence of fixed nitrogen on Azotobacter. J Bacteriol 19(6):389–414

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  91. Strandberg GW, Wilson PW (1968) Formation of the nitrogen-fixing enzyme system in Azotobacter vinelandii. Can J Microbiol 14(1):25–31. https://doi.org/10.1139/m68-005

    Article  CAS  PubMed  Google Scholar 

  92. Nagatani HH, Brill WJ (1974) Nitrogenase V. the effect of Mo, W and V on the synthesis of nitrogenase components in Azotobacter vinelandii. Biochim Biophys Acta 362(1):160–166. https://doi.org/10.1016/0304-4165(74)90037-3

    Article  CAS  PubMed  Google Scholar 

  93. Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227(5259):680–685. https://doi.org/10.1038/227680a0

    Article  CAS  PubMed  Google Scholar 

  94. Neuhoff V, Arold N, Taube D, Ehrhardt W (1988) Improved staining of proteins in polyacrylamide gels including isoelectric focusing gels with clear background at nanogram sensitivity using Coomassie brilliant blue G-250 and R-250. Electrophoresis 9(6):255–262. https://doi.org/10.1002/elps.1150090603

    Article  CAS  PubMed  Google Scholar 

  95. Kang D, Suh M-K et al (2002) Highly sensitive and fast protein detection with coomassie brilliant blue in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (highly sensitive and fast protein detection with coomassie brilliant blue in sodium dodecyl sulfate-polyacrylamide gel electrophoresis). Bull Korean Chem Soc 23:1511–1512. https://doi.org/10.5012/BKCS.2002.23.11.1511

    Article  CAS  Google Scholar 

  96. Smith PK, Krohn RI, Hermanson GT, Mallia AK, Gartner FH, Provenzano MD, Fujimoto EK, Goeke NM, Olson BJ, Klenk DC (1985) Measurement of protein using bicinchoninic acid. Anal Biochem 150(1):76–85. https://doi.org/10.1016/0003-2697(85)90442-7

    Article  CAS  PubMed  Google Scholar 

  97. Sippel D (2017) Structure of the vanadium Nitrogenase of Azotobacter vinelandii and mechanistic insights into biological nitrogen fixation, https://doi.org/10.6094/UNIFR/13383, Dissertation at the University of Freiburg

  98. Searle PL (1984) The berthelot or indophenol reaction and its use in the analytical chemistry of nitrogen. A review. Analyst 109(5):549–568. https://doi.org/10.1039/AN9840900549

    Article  CAS  Google Scholar 

  99. Eady RR, Richardson TH, Miller RW, Hawkins M, Lowe DJ (1988) The vanadium nitrogenase of Azotobacter chroococcum. Purification and properties of the Fe protein. Biochem J 256(1):189–196. https://doi.org/10.1042/bj2560189

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  100. Jasniewski AJ, Lee CC, Ribbe MW, Hu Y (2020) Reactivity, mechanism, and assembly of the alternative Nitrogenases. Chem Rev 120(12):5107–5157. https://doi.org/10.1021/acs.chemrev.9b00704

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Authors and Affiliations

  1. Institut für Biochemie, Albert-Ludwigs-Universität Freiburg, Freiburg, Germany

    Katharina Parison, Jakob Gies-Elterlein, Christian Trncik & Oliver Einsle

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  1. Katharina Parison
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  2. Jakob Gies-Elterlein
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  3. Christian Trncik
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  4. Oliver Einsle
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Correspondence to Oliver Einsle .

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  1. Department of Chemistry, Wake Forest University, Winston Salem, NC, USA

    Patricia C. Dos Santos

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Parison, K., Gies-Elterlein, J., Trncik, C., Einsle, O. (2021). Expression, Isolation, and Characterization of Vanadium Nitrogenase from Azotobacter vinelandii. In: Dos Santos, P.C. (eds) Fe-S Proteins. Methods in Molecular Biology, vol 2353. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1605-5_6

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