• Nathaniel S. Sickerman
  • Yilin HuEmail author
  • Markus W. RibbeEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1876)


Biological nitrogen fixation, the conversion of dinitrogen (N2) into ammonia (NH3), stands as a particularly challenging chemical process. As the entry point into a bioavailable form of nitrogen, biological nitrogen fixation is a critical step in the global nitrogen cycle. In Nature, only one enzyme, nitrogenase, is competent in performing this reaction. Study of this complex metalloenzyme has revealed a potent substrate reduction system that utilizes some of the most sophisticated metalloclusters known. This chapter discusses the structure and function of nitrogenase, covers methods that have proven useful in the elucidation of enzyme properties, and provides an overview of the three known nitrogenase variants.

Key words

Biological nitrogen fixation Nitrogenase MoFe protein Fe protein P-cluster M-cluster 



The authors are supported by the National Institutes of Health grant GM67626 (to M.W.R. and Y.H.).


  1. 1.
    Brill WJ (1980) Biochemical genetics of nitrogen fixation. Microbiol Rev 44:449–467PubMedPubMedCentralGoogle Scholar
  2. 2.
    Howard JB, Rees DC (1996) Structural basis of biological nitrogen fixation. Chem Rev 96:2965–2982CrossRefGoogle Scholar
  3. 3.
    Burgess BK, Lowe DJ (1996) Mechanism of molybdenum nitrogenase. Chem Rev 96:2983–3011CrossRefGoogle Scholar
  4. 4.
    Lee CC, Hu Y, Ribbe MW (2010) Vanadium nitrogenase reduces CO. Science 329:642–642PubMedPubMedCentralCrossRefGoogle Scholar
  5. 5.
    Hardy RWF, Knight E (1967) ATP-dependent reduction of azide and HCN by N2-fixing enzymes of Azotobacter vinelandii and Clostridium pasteurianum. Biochim Biophys Acta 139:69–90PubMedCrossRefPubMedCentralGoogle Scholar
  6. 6.
    Dilworth MJ (1966) Acetylene reduction by nitrogen-fixing preparations from Clostridium pasteurianum. Biochim Biophys Acta 127:285–294PubMedCrossRefPubMedCentralGoogle Scholar
  7. 7.
    Seefeldt LC, Rasche ME, Ensign SA (1995) Carbonyl sulfide and carbon dioxide as new substrates, and carbon disulfide as a new inhibitor, of nitrogenase. Biochemistry 34:5382–5389PubMedCrossRefPubMedCentralGoogle Scholar
  8. 8.
    Joerger RD, Bishop PE (1988) Bacterial alternative nitrogen fixation systems. Crit Rev Microbiol 16:1–14PubMedCrossRefPubMedCentralGoogle Scholar
  9. 9.
    Eady RR (1996) Structure-function relationships of alternative nitrogenases. Chem Rev 96:3013–3030PubMedCrossRefPubMedCentralGoogle Scholar
  10. 10.
    Jacobson MR, Brigle KE, Bennett LT et al (1989) Physical and genetic map of the major nif gene cluster from Azotobacter vinelandii. J Bacteriol 171:1017–1027PubMedPubMedCentralCrossRefGoogle Scholar
  11. 11.
    Robinson AC, Dean DR, Burgess BK (1987) Iron-molybdenum cofactor biosynthesis in Azotobacter vinelandii requires the iron protein of nitrogenase. J Biol Chem 262:14327–14332PubMedPubMedCentralGoogle Scholar
  12. 12.
    Ribbe MW, Hu Y, Guo M et al (2002) The FeMoco-deficient MoFe protein produced by a nifH deletion strain of Azotobacter vinelandii shows unusual P-cluster features. J Biol Chem 277:23469–23476PubMedCrossRefPubMedCentralGoogle Scholar
  13. 13.
    Hu Y, Fay AW, Lee CC et al (2007) P-cluster maturation on nitrogenase MoFe protein. Proc Natl Acad Sci U S A 104:10424–10429PubMedPubMedCentralCrossRefGoogle Scholar
  14. 14.
    Hu Y, Corbett MC, Fay AW et al (2006) Nitrogenase Fe protein: a molybdate/homocitrate insertase. Proc Natl Acad Sci U S A 103:17125–17130PubMedPubMedCentralCrossRefGoogle Scholar
  15. 15.
    Georgiadis MM, Komiya H, Chakrabarti P et al (1992) Crystallographic structure of the nitrogenase iron protein from Azotobacter vinelandii. Science 257:1653–1659CrossRefGoogle Scholar
  16. 16.
    Lindahl PA, Day EP, Kent TA et al (1985) Moessbauer, EPR, and magnetization studies of the Azotobacter vinelandii iron protein. Evidence for a [4Fe4S]1+ cluster with spin S = 3/2. J Biol Chem 260:11160–11173PubMedPubMedCentralGoogle Scholar
  17. 17.
    Lanzilotta WN, Ryle MJ, Seefeldt LC (1995) Nucleotide hydrolysis and protein conformational changes in Azotobacter vinelandii nitrogenase iron protein: defining the function of aspartate 129. Biochemistry 34:10713–10723PubMedCrossRefPubMedCentralGoogle Scholar
  18. 18.
    Watt GD, Reddy KRN (1994) Formation of an all ferrous Fe4S4 cluster in the iron protein component of Azotobacter vinelandii nitrogenase. J Inorg Biochem 53:281–294CrossRefGoogle Scholar
  19. 19.
    Angove HC, Yoo SJ, Burgess BK et al (1997) Mössbauer and EPR Evidence for an all-ferrous Fe4S4 cluster with S = 4 in the Fe protein of nitrogenase. J Am Chem Soc 119:8730–8731CrossRefGoogle Scholar
  20. 20.
    Angove HC, Yoo SJ, Munck E et al (1998) An all-ferrous state of the Fe protein of nitrogenase: interaction with nucleotides and electron transfer to the MoFe protein. J Biol Chem 273:26330–26337PubMedCrossRefPubMedCentralGoogle Scholar
  21. 21.
    Vincent KA, Tilley GJ, Quammie NC et al (2003) Instantaneous, stoichiometric generation of powerfully reducing states of protein active sites using Eu(II) and polyaminocarboxylate ligands. Chem Commun 20:2590–2591CrossRefGoogle Scholar
  22. 22.
    Lowery TJ, Wilson PE, Zhang B et al (2006) Flavodoxin hydroquinone reduces Azotobacter vinelandii Fe protein to the all-ferrous redox state with a S = 0 spin state. Proc Natl Acad Sci U S A 103:17131–17136PubMedPubMedCentralCrossRefGoogle Scholar
  23. 23.
    Johnson JL, Tolley AM, Erickson JA et al (1996) Steady-state kinetic studies of dithionite utilization, component protein interaction, and the formation of an oxidized iron protein intermediate during Azotobacter vinelandii nitrogenase catalysis. Biochemistry 35:11336–11342PubMedCrossRefPubMedCentralGoogle Scholar
  24. 24.
    Morgan TV, Mortenson LE, McDonald JW et al (1988) Comparison of redox and EPR properties of the molybdenum iron proteins of Clostridium pasteurianum and Azotobacter vinelandii nitrogenases. J Inorg Biochem 33:111–120PubMedCrossRefPubMedCentralGoogle Scholar
  25. 25.
    Corbett MC, Hu Y, Fay AW et al (2007) Conformational differences between Azotobacter vinelandii nitrogenase MoFe proteins as studied by small-angle X-ray scattering. Biochemistry 46:8066–8074PubMedCrossRefPubMedCentralGoogle Scholar
  26. 26.
    Lanzilotta WN, Parker VD, Seefeldt LC (1999) Thermodynamics of nucleotide interactions with the Azotobacter vinelandii nitrogenase iron protein. Biochim Biophys Acta 1429:411–421PubMedCrossRefPubMedCentralGoogle Scholar
  27. 27.
    Sarma R, Mulder DW, Brecht E et al (2007) Probing the MgATP-bound conformation of the nitrogenase Fe protein by solution small-angle X-ray scattering. Biochemistry 46:14058–14066PubMedPubMedCentralCrossRefGoogle Scholar
  28. 28.
    Schlessman JL, Woo D, Joshua-Tor L et al (1998) Conformational variability in structures of the nitrogenase iron proteins from Azotobacter vinelandii and Clostridium pasteurianum. J Mol Biol 280:669–685CrossRefGoogle Scholar
  29. 29.
    Kim J, Rees DC (1992) Crystallographic structure and functional implications of the nitrogenase molybdenum-iron protein from Azotobacter vinelandii. Nature 360:553–560CrossRefGoogle Scholar
  30. 30.
    Schindelin H, Kisker C, Schlessman JL et al (1997) Structure of ADP·AlF4-stabilized nitrogenase complex and its implications for signal transduction. Nature 387:370–376CrossRefGoogle Scholar
  31. 31.
    Kaiser JT, Hu Y, Wiig JA et al (2011) Structure of precursor-bound NifEN: a nitrogenase FeMo cofactor maturase/insertase. Science 331:91–94PubMedPubMedCentralCrossRefGoogle Scholar
  32. 32.
    Maier RJ, Moshiri F (2000) Role of the Azotobacter vinelandii nitrogenase-protective Shethna protein in preventing oxygen-mediated cell death. J Bacteriol 182:3854–3857PubMedPubMedCentralCrossRefGoogle Scholar
  33. 33.
    Chan MK, Kim J, Rees DC (1993) The nitrogenase iron-molybdenum-cofactor and P-cluster pair: 2.2 Å resolution structures. Science 260:792–794PubMedCrossRefGoogle Scholar
  34. 34.
    Pierik AJ, Wassink H, Haaker H et al (1993) Redox properties and EPR spectroscopy of the P clusters of Azotobacter vinelandii molybdenum-iron protein. Eur J Biochem 212:51–61PubMedCrossRefGoogle Scholar
  35. 35.
    Huynh BH, Henzl MT, Christner JA et al (1980) Nitrogenase XII. Mössbauer studies of the MoFe protein from Clostridium pasteurianum W5. Biochim Biophys Acta 623:124–138PubMedCrossRefPubMedCentralGoogle Scholar
  36. 36.
    Peters JW, Stowell MHB, Soltis SM et al (1997) Redox-dependent structural changes in the nitrogenase P-cluster. Biochemistry 36:1181–1187PubMedPubMedCentralCrossRefGoogle Scholar
  37. 37.
    Surerus KK, Hendrich MP, Christie PD et al (1992) Moessbauer and integer-spin EPR of the oxidized P-clusters of nitrogenase: POX is a non-Kramers system with a nearly degenerate ground doublet. J Am Chem Soc 114:8579–8590CrossRefGoogle Scholar
  38. 38.
    Rupnik K, Hu Y, Lee CC et al (2012) P+ State of nitrogenase P-cluster exhibits electronic structure of a [Fe4S4]+ cluster. J Am Chem Soc 134:13749–13754PubMedPubMedCentralCrossRefGoogle Scholar
  39. 39.
    Einsle O, Tezcan FA, Andrade SLA et al (2002) Nitrogenase MoFe-protein at 1.16 Å resolution: a central ligand in the FeMo-cofactor. Science 297:1696–1700CrossRefGoogle Scholar
  40. 40.
    Spatzal T, Aksoyoglu M, Zhang L et al (2011) Evidence for interstitial carbon in nitrogenase FeMo cofactor. Science 334:940–940PubMedPubMedCentralCrossRefGoogle Scholar
  41. 41.
    Wiig JA, Hu Y, Lee CC et al (2012) Radical SAM-dependent carbon insertion into the nitrogenase M-cluster. Science 337:1672–1675PubMedCrossRefPubMedCentralGoogle Scholar
  42. 42.
    Wiig JA, Hu Y, Ribbe MW (2015) Refining the pathway of carbide insertion into the nitrogenase M-cluster. Nat Commun 6:8034PubMedPubMedCentralCrossRefGoogle Scholar
  43. 43.
    Shah VK, Brill WJ (1977) Isolation of an iron-molybdenum cofactor from nitrogenase. Proc Natl Acad Sci U S A 74:3249–3253PubMedPubMedCentralCrossRefGoogle Scholar
  44. 44.
    McLean PA, Wink DA, Chapman SK et al (1989) A new method for extraction of iron-molybdenum cofactor (FeMoco) from nitrogenase adsorbed to DEAE-cellulose. 1. Effects of anions, cations, and preextraction treatments. Biochemistry 28:9402–9406PubMedCrossRefPubMedCentralGoogle Scholar
  45. 45.
    Christiansen J, Goodwin PJ, Lanzilotta WN et al (1998) Catalytic and biophysical properties of a nitrogenase apo-MoFe protein produced by a nifB-deletion mutant of Azotobacter vinelandii. Biochemistry 37:12611–12623PubMedCrossRefGoogle Scholar
  46. 46.
    Lee CC, Hu Y, Ribbe MW (2012) ATP-independent formation of hydrocarbons catalyzed by isolated nitrogenase cofactors. Angew Chem Int Ed 51:1947–1949CrossRefGoogle Scholar
  47. 47.
    Lee CC, Hu Y, Ribbe MW (2015) Catalytic reduction of CN, CO, and CO2 by nitrogenase cofactors in lanthanide-driven reactions. Angew Chem Int Ed 54:1219–1222CrossRefGoogle Scholar
  48. 48.
    Tanifuji K, Sickerman N, Lee CC et al (2016) Structure and reactivity of an asymmetric synthetic mimic of nitrogenase cofactor. Angew Chem Int Ed 55:15633–15636CrossRefGoogle Scholar
  49. 49.
    Igarashi RY, Dos Santos PC, Niehaus WG et al (2004) Localization of a catalytic intermediate bound to the FeMo-cofactor of nitrogenase. J Biol Chem 279:34770–34775PubMedCrossRefGoogle Scholar
  50. 50.
    Brigle KE, Setterquist RA, Dean DR et al (1987) Site-directed mutagenesis of the nitrogenase MoFe protein of Azotobacter vinelandii. Proc Natl Acad Sci U S A 84:7066–7069PubMedPubMedCentralCrossRefGoogle Scholar
  51. 51.
    Dilworth MJ, Fisher K, Kim C-H et al (1998) Effects on substrate reduction of substitution of histidine-195 by glutamine in the α-subunit of the MoFe protein of Azotobacter vinelandii nitrogenase. Biochemistry 37:17495–17505PubMedCrossRefPubMedCentralGoogle Scholar
  52. 52.
    Fisher K, Dilworth MJ, Kim C-H et al (2000) Azotobacter vinelandii nitrogenases containing altered MoFe proteins with substitutions in the FeMo-cofactor environment: effects on the catalyzed reduction of acetylene and ethylene. Biochemistry 39:2970–2979PubMedCrossRefPubMedCentralGoogle Scholar
  53. 53.
    Orme-Johnson WH, Hamilton WD, Jones TL et al (1972) Electron paramagnetic resonance of nitrogenase and nitrogenase components from Clostridium pasteurianum W5 and Azotobacter vinelandii OP. Proc Natl Acad Sci U S A 69:3142–3145PubMedPubMedCentralCrossRefGoogle Scholar
  54. 54.
    O'Donnell MJ, Smith BE (1978) Electron-paramagnetic-resonance studies on the redox properties of the molybdenum-iron protein of nitrogenase between +50 and −450 mV. Biochem J 173:831–838PubMedPubMedCentralCrossRefGoogle Scholar
  55. 55.
    Watt GD, Burns A, Lough S et al (1980) Redox and spectroscopic properties of oxidized MoFe protein from Azotobacter vinelandii. Biochemistry 19:4926–4932PubMedCrossRefPubMedCentralGoogle Scholar
  56. 56.
    Yoo SJ, Angove HC, Papaefthymiou V et al (2000) Mössbauer study of the MoFe protein of nitrogenase from Azotobacter vinelandii using selective 57Fe enrichment of the M-centers. J Am Chem Soc 122:4926–4936CrossRefGoogle Scholar
  57. 57.
    Bjornsson R, Lima FA, Spatzal T et al (2014) Identification of a spin-coupled Mo(III) in the nitrogenase iron-molybdenum cofactor. Chem Sci 5:3096–3103CrossRefGoogle Scholar
  58. 58.
    Spatzal T, Schlesier J, Burger EM et al (2016) Nitrogenase FeMoco investigated by spatially resolved anomalous dispersion refinement. Nat Commun 7:10902PubMedPubMedCentralCrossRefGoogle Scholar
  59. 59.
    Tezcan FA, Kaiser JT, Mustafi D et al (2005) Nitrogenase complexes: multiple docking sites for a nucleotide switch protein. Science 309:1377–1380CrossRefGoogle Scholar
  60. 60.
    Chiu H-J, Peters JW, Lanzilotta WN et al (2001) MgATP-bound and nucleotide-free structures of a nitrogenase protein complex between the Leu 127Δ-Fe-protein and the MoFe-protein. Biochemistry 40:641–650PubMedCrossRefPubMedCentralGoogle Scholar
  61. 61.
    Schmid B, Einsle O, Chiu H-J et al (2002) Biochemical and structural characterization of the cross-linked complex of nitrogenase: comparison to the ADP-AlF4-stabilized structure. Biochemistry 41:15557–15565CrossRefGoogle Scholar
  62. 62.
    Emerich DW, Burris RH (1976) Interactions of heterologous nitrogenase components that generate catalytically inactive complexes. Proc Natl Acad Sci U S A 73:4369–4373PubMedPubMedCentralCrossRefGoogle Scholar
  63. 63.
    Emerich DW, Ljones T, Burris RH (1978) Nitrogenase: properties of the catalytically inactive complex between the Azotobacter vinelandii MoFe protein and the Clostridium pasteurianum Fe protein. Biochim Biophys Acta 527:359–369PubMedCrossRefPubMedCentralGoogle Scholar
  64. 64.
    Emerich DW, Burris RH (1978) Complementary functioning of the component proteins of nitrogenase from several bacteria. J Bacteriol 134:936–943PubMedPubMedCentralGoogle Scholar
  65. 65.
    Chan JM, Ryle MJ, Seefeldt LC (1999) Evidence that MgATP accelerates primary electron transfer in a Clostridium pasteurianum Fe protein-Azotobacter vinelandii MoFe protein nitrogenase tight complex. J Biol Chem 274:17593–17598PubMedCrossRefPubMedCentralGoogle Scholar
  66. 66.
    Danyal K, Dean DR, Hoffman BM et al (2011) Electron transfer within nitrogenase: evidence for a deficit-spending mechanism. Biochemistry 50:9255–9263PubMedPubMedCentralCrossRefGoogle Scholar
  67. 67.
    Danyal K, Mayweather D, Dean DR et al (2010) Conformational gating of electron transfer from the nitrogenase Fe protein to MoFe protein. J Am Chem Soc 132:6894–6895PubMedPubMedCentralCrossRefGoogle Scholar
  68. 68.
    Yang ZY, Ledbetter R, Shaw S et al (2016) Evidence that the Pi release event is the rate-limiting step in the nitrogenase catalytic cycle. Biochemistry 55:3625–3635PubMedCrossRefGoogle Scholar
  69. 69.
    Thorneley RNF, Lowe DJ (1984) The mechanism of Klebsiella pneumoniae nitrogenase action. Pre-steady-state kinetics of an enzyme-bound intermediate in nitrogen reduction and of ammonia formation. Biochem J 224:887–894PubMedPubMedCentralCrossRefGoogle Scholar
  70. 70.
    Thorneley RN, Lowe DJ (1984) The mechanism of Klebsiella pneumoniae nitrogenase action. Pre-steady-state kinetics of an enzyme-bound intermediate in N2 reduction and of NH3 formation. Biochem J 224:887–894PubMedPubMedCentralCrossRefGoogle Scholar
  71. 71.
    Thorneley RNF, Lowe DJ (1984) The mechanism of Klebsiella pneumoniae nitrogenase action. Simulation of the dependences of H2-evolution rate on component-protein concentration and ratio and sodium dithionite concentration. Biochem J 224:903PubMedPubMedCentralCrossRefGoogle Scholar
  72. 72.
    Lee HI, Sørlie M, Christiansen J et al (2005) Electron inventory, kinetic assignment (En), structure, and bonding of nitrogenase turnover intermediates with C2H2 and CO. J Am Chem Soc 127:15880–15890PubMedCrossRefPubMedCentralGoogle Scholar
  73. 73.
    Hoffman BM, Lukoyanov D, Yang Z-Y et al (2014) Mechanism of nitrogen fixation by nitrogenase: the next stage. Chem Rev 114:4041–4062PubMedPubMedCentralCrossRefGoogle Scholar
  74. 74.
    Hoffman BM (1994) ENDOR and ESEEM of a non-Kramers doublet in an integer-spin system. J Phys Chem 98:11657–11665CrossRefGoogle Scholar
  75. 75.
    Pollock RC, Lee H-I, Cameron LM et al (1995) Investigation of CO bound to inhibited forms of nitrogenase MoFe protein by 13C ENDOR. J Am Chem Soc 117:8686–8687CrossRefGoogle Scholar
  76. 76.
    Christie PD, Lee H-I, Cameron LM et al (1996) Identification of the CO-binding cluster in nitrogenase MoFe protein by ENDOR of 57Fe isotopomers. J Am Chem Soc 118:8707–8709CrossRefGoogle Scholar
  77. 77.
    Lee H-I, Sørlie M, Christiansen J et al (2000) Characterization of an intermediate in the reduction of acetylene by the nitrogenase α-Gln195 MoFe protein by Q-band EPR and 13C,1H ENDOR. J Am Chem Soc 122:5582–5587CrossRefGoogle Scholar
  78. 78.
    Seefeldt LC, Hoffman BM, Dean DR (2009) Mechanism of Mo-dependent nitrogenase. Annu Rev Biochem 78:701–722PubMedPubMedCentralCrossRefGoogle Scholar
  79. 79.
    Lukoyanov D, Khadka N, Yang Z-Y et al (2016) Reductive elimination of H2 activates nitrogenase to reduce the N≡N triple bond: characterization of the E4(4H) Janus nitermediate in wild-type enzyme. J Am Chem Soc 138:10674–10683PubMedPubMedCentralCrossRefGoogle Scholar
  80. 80.
    Chatt J, Pearman AJ, Richards RL (1975) Diazenido (iminonitrosyl) (N2H), hydrazido(2) (N2H2), and hydrazido(1-) (N2H3) ligands as intermediates in the reduction of ligating dinitrogen to ammonia. J Organomet Chem 101:C45–C47CrossRefGoogle Scholar
  81. 81.
    Chatt J, Pearman AJ, Richards RL (1975) Reduction of monocoordinated molecular nitrogen to ammonia in a protic environment. Nature 253:39–40CrossRefGoogle Scholar
  82. 82.
    Chatt J, Pearman AJ, Richards RL (1976) Relevance of oxygen ligands to reduction of ligating dinitrogen. Nature 259:204PubMedCrossRefPubMedCentralGoogle Scholar
  83. 83.
    Chatt J, Pearman AJ, Richards RL (1977) Conversion of dinitrogen in its molybdenum and tungsten complexes into ammonia and possible relevance to the nitrogenase reaction. J Chem Soc Dalton Trans 19:1852–1860Google Scholar
  84. 84.
    Yandulov DV, Schrock RR (2002) Reduction of dinitrogen to ammonia at a well-protected reaction site in a molybdenum triamidoamine complex. J Am Chem Soc 124:6252–6253PubMedCrossRefPubMedCentralGoogle Scholar
  85. 85.
    Yandulov DV, Schrock RR (2003) Catalytic reduction of dinitrogen to ammonia at a single molybdenum center. Science 301:76–78PubMedCrossRefPubMedCentralGoogle Scholar
  86. 86.
    Siemann S, Schneider K, Dröttboom M et al (2002) The Fe-only nitrogenase and the Mo nitrogenase from Rhodobacter capsulatus. Eur J Biochem 269:1650–1661PubMedCrossRefPubMedCentralGoogle Scholar
  87. 87.
    Davis R, Lehman L, Petrovich R et al (1996) Purification and characterization of the alternative nitrogenase from the photosynthetic bacterium Rhodospirillum rubrum. J Bacteriol 178:1445–1450PubMedPubMedCentralCrossRefGoogle Scholar
  88. 88.
    Anderson JS, Cutsail GE 3rd, Rittle J et al (2015) Characterization of an Fe≡N–NH2 intermediate relevant to catalytic N2 reduction to NH3. J Am Chem Soc 137:7803–7809PubMedPubMedCentralCrossRefGoogle Scholar
  89. 89.
    Rittle J, Peters JC (2016) An Fe-N2 complex that generates hydrazine and ammonia via Fe═NNH2: demonstrating a hybrid distal-to-alternating pathway for N2 reduction. J Am Chem Soc 138:4243–4248PubMedPubMedCentralCrossRefGoogle Scholar
  90. 90.
    Dance I (2014) A unified chemical mechanism for hydrogenation reactions catalyzed by nitrogenase. In: Bioinspired Catalysis. Wiley-VCH Verlag GmbH & Co, KGaA, pp 249–288Google Scholar
  91. 91.
    Dilworth MJ, Eady RR, Robson RL et al (1987) Ethane formation from acetylene as a potential test for vanadium nitrogenase in vivo. Nature 327:167–168CrossRefGoogle Scholar
  92. 92.
    Müller A, Schneider K, Gollan U et al (1995) Characterization of the “iron only” nitrogenase from Rhodobacter capsulatus. J Inorg Biochem 59:551CrossRefGoogle Scholar
  93. 93.
    Hu Y, Lee CC, Ribbe MW (2011) Extending the carbon chain: hydrocarbon formation catalyzed by vanadium/molybdenum nitrogenases. Science 333:753–755PubMedPubMedCentralCrossRefGoogle Scholar
  94. 94.
    Hwang JC, Chen CH, Burris RH (1973) Inhibition of nitrogenase-catalyzed reductions. Biochim Biophys Acta 292:256–270PubMedCrossRefPubMedCentralGoogle Scholar
  95. 95.
    Walmsley J, Toukdarian A, Kennedy C (1994) The role of regulatory genes nifA, vnfA, anfA, nfrX, ntrC, and rpoN in expression of genes encoding the three nitrogenases of Azotobacter vinelandii. Arch Microbiol 162:422–429PubMedCrossRefPubMedCentralGoogle Scholar
  96. 96.
    Kennedy C, Dean D (1992) The nifU, nifS and nifV gene products are required for activity of all three nitrogenases of Azotobacter vinelandii. Mol Gen Genet 231:494–498PubMedCrossRefPubMedCentralGoogle Scholar
  97. 97.
    Kennedy C, Gamal R, Humphrey R et al (1986) The nifH, nifM and nifN genes of Azotobacter vinelandii: characterisation by Tn5 mutagenesis and isolation from pLAFR1 gene banks. Mol Gen Genet 205:318–325CrossRefGoogle Scholar
  98. 98.
    Paul W, Merrick M (1989) The roles of the nifW, nifZ, and nifM genes of Klebsiella pneumoniae in nitrogenase biosynthesis. Eur J Biochem 178:675–682PubMedCrossRefPubMedCentralGoogle Scholar
  99. 99.
    Jacobson MR, Cash VL, Weiss MC et al (1989) Biochemical and genetic analysis of the nifUSVWZM cluster from Azotobacter vinelandii. Mol Gen Genet 219:49–57PubMedCrossRefPubMedCentralGoogle Scholar
  100. 100.
    Ribbe MW, Hu Y, Hodgson KO et al (2014) Biosynthesis of nitrogenase metalloclusters. Chem Rev 114:4063–4080PubMedCrossRefPubMedCentralGoogle Scholar
  101. 101.
    Zheng L, White RH, Cash VL et al (1994) Mechanism for the desulfurization of L-cysteine catalyzed by the nifS gene product. Biochemistry 33:4714–4720PubMedCrossRefPubMedCentralGoogle Scholar
  102. 102.
    Yuvaniyama P, Agar JN, Cash VL et al (2000) NifS-directed assembly of a transient [2Fe-2S] cluster within the NifU protein. Proc Natl Acad Sci U S A 97:599–604PubMedPubMedCentralCrossRefGoogle Scholar
  103. 103.
    Smith AD, Jameson GN, Dos Santos PC et al (2005) NifS-mediated assembly of [4Fe-4S] clusters in the N- and C-terminal domains of the NifU scaffold protein. Biochemistry 44:12955–12969PubMedCrossRefPubMedCentralGoogle Scholar
  104. 104.
    Zheng L, White RH, Dean DR (1997) Purification of the Azotobacter vinelandii nifV-encoded homocitrate synthase. J Bacteriol 179:5963–5966PubMedPubMedCentralCrossRefGoogle Scholar
  105. 105.
    Wiig JA, Lee CC, Hu Y et al (2013) Tracing the interstitial carbide of the nitrogenase cofactor during substrate turnover. J Am Chem Soc 135:4982–4983PubMedPubMedCentralCrossRefGoogle Scholar
  106. 106.
    Fay AW, Wiig JA, Lee CC et al (2015) Identification and characterization of functional homologs of nitrogenase cofactor biosynthesis protein NifB from methanogens. Proc Natl Acad Sci U S A 112:14829–14833PubMedPubMedCentralCrossRefGoogle Scholar
  107. 107.
    Kennedy C, Bali A, Blanco G et al (1991) Regulation of expression of genes for three nitrogenases in Azotobacter vinelandii. In: Polsinelli M, Materassi R, Vincenzini M (eds) Developments in plant and soil sciences, Nitrogen fixation, vol 48. Springer Netherlands, Dordrecht, pp 13–23Google Scholar
  108. 108.
    Rebelein JG, Stiebritz MT, Lee CC et al (2017) Activation and reduction of carbon dioxide by nitrogenase iron proteins. Nat Chem Biol 13:147–149PubMedCrossRefPubMedCentralGoogle Scholar
  109. 109.
    Blank MA, Lee CC, Hu Y et al (2011) Structural models of the [Fe4S4] clusters of homologous nitrogenase Fe proteins. Inorg Chem 50:7123–7128PubMedPubMedCentralCrossRefGoogle Scholar
  110. 110.
    Robson RL, Woodley PR, Pau RN et al (1989) Structural genes for the vanadium nitrogenase from Azotobacter chroococcum. EMBO J 8:1217–1224PubMedPubMedCentralCrossRefGoogle Scholar
  111. 111.
    Fallik E, Robson RL (1990) Completed sequence of the region encoding the structural genes for the vanadium nitrogenase of Azotobacter chroococcum. Nucleic Acids Res 18:4616PubMedPubMedCentralCrossRefGoogle Scholar
  112. 112.
    Sippel D, Schlesier J, Rohde M et al (2017) Production and isolation of vanadium nitrogenase from Azotobacter vinelandii by molybdenum depletion. J Biol Inorg Chem 22:161–168PubMedCrossRefPubMedCentralGoogle Scholar
  113. 113.
    Lee CC, Hu Y, Ribbe MW (2009) Unique features of the nitrogenase VFe protein from Azotobacter vinelandii. Proc Natl Acad Sci U S A 106:9209–9214PubMedPubMedCentralCrossRefGoogle Scholar
  114. 114.
    Ravi N, Moore V, Lloyd SG et al (1994) Mössbauer characterization of the metal clusters in Azotobacter vinelandii nitrogenase VFe protein. J Biol Chem 269:20920–20924PubMedPubMedCentralGoogle Scholar
  115. 115.
    Eady RR, Richardson TH, Miller RW et al (1988) The vanadium nitrogenase of Azotobacter chroococcum. Purification and properties of the iron protein. Biochem J 256:189–196PubMedPubMedCentralCrossRefGoogle Scholar
  116. 116.
    Smith BE, Eady RR, Lowe DJ et al (1988) The vanadium-iron protein of vanadium nitrogenase from Azotobacter chroococcum contains an iron-vanadium cofactor. Biochem J 250:299–302PubMedPubMedCentralCrossRefGoogle Scholar
  117. 117.
    Arber JM, Dobson BR, Eady RR et al (1987) Vanadium K-edge X-ray absorption spectrum of the VFe protein of the vanadium nitrogenase of Azotobacter chroococcum. Nature 325:372–374CrossRefGoogle Scholar
  118. 118.
    Arber JM, Dobson BR, Eady RR et al (1989) Vanadium K-edge X-ray-absorption spectroscopy of the functioning and thionine-oxidized forms of the vanadium-iron protein of the vanadium nitrogenase from Azotobacter chroococcum. Biochem J 258:733–737PubMedPubMedCentralCrossRefGoogle Scholar
  119. 119.
    Harvey I, Arber JM, Eady RR et al (1990) Iron K-edge X-ray-absorption spectroscopy of the iron-vanadium cofactor of the vanadium nitrogenase from Azotobacter chroococcum. Biochem J 266:929–931PubMedPubMedCentralGoogle Scholar
  120. 120.
    Rees JA, Bjornsson R, Schlesier J et al (2015) The Fe–V cofactor of vanadium nitrogenase contains an Interstitial carbon atom. Angew Chem Int Ed 54:13249–13252CrossRefGoogle Scholar
  121. 121.
    Lee CC, Fay AW, Weng TC et al (2015) Uncoupling binding of substrate CO from turnover by vanadium nitrogenase. Proc Natl Acad Sci U S A 112:13845–13849PubMedPubMedCentralCrossRefGoogle Scholar
  122. 122.
    George GN, Coyle CL, Hales BJ et al (1988) X-ray absorption of Azotobacter vinelandii vanadium nitrogenase. J Am Chem Soc 110:4057–4059CrossRefGoogle Scholar
  123. 123.
    Fay AW, Blank MA, Lee CC et al (2010) Characterization of isolated nitrogenase FeVco. J Am Chem Soc 132:12612–12618PubMedPubMedCentralCrossRefGoogle Scholar
  124. 124.
    Sippel D, Einsle O (2017) The structure of vanadium nitrogenase reveals an unusual bridging ligand. Nat Chem Biol 13:956–960PubMedPubMedCentralCrossRefGoogle Scholar
  125. 125.
    Schloegl R (2003) Catalytic synthesis of ammonia–a "never-ending story"? Angew Chem Int Ed 42:2004–2008CrossRefGoogle Scholar
  126. 126.
    Cherkasov N, Ibhadon AO, Fitzpatrick P (2015) A review of the existing and alternative methods for greener nitrogen fixation. Chem Eng Process 90:24–33CrossRefGoogle Scholar
  127. 127.
    Hu Y, Fay AW, Ribbe MW (2005) Identification of a nitrogenase FeMo cofactor precursor on NifEN complex. Proc Natl Acad Sci U S A 102:3236–3241PubMedPubMedCentralCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Department of Molecular Biology and BiochemistryUniversity of California, IrvineIrvineUSA
  2. 2.Department of ChemistryUniversity of California, IrvineIrvineUSA

Personalised recommendations