Assay Methods for Products of Nitrogenase Action on Substrates

  • M. J. Dilworth
Part of the Nitrogen Fixation: Origins, Applications, and Research Progress book series (NITR, volume 1)


Systems for the steady-state assay of the molybdenum-, vanadium-, and iron-only nitrogenases share a number of common requirements.


Propargyl Alcohol Carbonyl Sulfide MoFe Protein Azotobacter Chroococcum Clostridium Pasteurianum 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. Ashby, G. A., Dilworth, M. J., and Thorneley, R. N. F. (1987). Klebsiella pneumoniae nitrogenase. Inhibition of hydrogen evolution by ethylene and the reduction of ethylene to ethane. Biochem. J., 247, 547–554.Google Scholar
  2. Beecher, G.R., and Whitten, G.R. (1970). Ammonia determination: Reagent modification and interfering compounds. Anal. Biochem., 36, 243–246.CrossRefGoogle Scholar
  3. Bonam, D., Murrell, S. A., and Ludden, P. W. (1984). Carbon monoxide dehydrogenase from Rhodospirillum rubrum. J. Bacteriol., 159, 693–699.Google Scholar
  4. Brune, M, Hunter, J. L., Corrie, J. E., and Webb, M. R. (1994). Direct, real-time measurement of rapid inorganic phosphate release using a novel fluorescent probe and its application to actomyosin subfragment 1 ATP»ase. Biochemistry, 33, 8262–8271.CrossRefGoogle Scholar
  5. Bulen, W. A. (1976). Nitrogenase from Azotobacter vinelandii and reactions affecting mechanistic interpretations. In W. E. Newton and C. J. Nyman (Eds.), Proc. First Internatl. Symp. Nitrogen Fixation(pp. 177–186). Pullman: Washington State University Press.Google Scholar
  6. Burgess, B. K., Wherland, S., Newton, W. E., and Stiefel, E. I. (1981). Nitrogenase reactivity: Insight into the nitrogen-fixing process through hydrogen-inhibition and HD-forming reactions. Biochemistry, 20, 5140–5146.CrossRefGoogle Scholar
  7. Burns, R. C, and Hardy, R. W. F. (1975). Nitrogen Fixation in Bacteria and Higher Plants, p. 132. Berlin: Springer-Verlag.CrossRefGoogle Scholar
  8. Burns, R. C, Hardy, R. W. F., and Phillips, W. D. (1975). Azotobacter nitrogenase: Mechanism and kinetics of allene reduction. In W. D. P. Stewart (Ed.), Nitrogen Fixation in Free-living Microorganisms (pp. 447–452). Cambridge: Cambridge University Press.Google Scholar
  9. Burris, R. H. (1972). Nitrogen fixation - assay methods and techniques. Methods Enzymol., 24B, 415–431.CrossRefGoogle Scholar
  10. Chaykin, S. (1969). Assay of nicotinamide deamidase. Determination of ammonia by the indophenol reaction. Anal. Biochem., 31, 375–382.CrossRefGoogle Scholar
  11. Clusius, K., and Hiirzeler, H. (1953). Reactions with nitrogen15. X. Reduction and oxidation of hydrazoic acid. Helv. Chim. Acta, 36, 1326–1332.CrossRefGoogle Scholar
  12. Conway, E. J. (1960). Microdiffusion Analysis and Volumetric Error. London: Crosby, Lock and Sons Ltd.Google Scholar
  13. Corbin, J. L. (1984). Liquid chromatographic-fluorescence determination of ammonia from nitrogenase reactions: A 2-min assay. Appl. Environ. Microbiol., 47, 1027–1030.Google Scholar
  14. Davis, L.C. (1980). Hydrazine as a substrate and inhibitor of Azotobacter vinelandii nitrogenase. Arch. Biochem. Biophys., 204, 270–276.CrossRefGoogle Scholar
  15. Dilworth, M. J. (1966). Acetylene reduction by nitrogen-fixing preparations from Clostridium pasteurianum. Biochim. Biophys. Acta, 127, 285–294.CrossRefGoogle Scholar
  16. Dilworth, M. J., and Eady, R. R. (1991). Hydrazine is a product of dinitrogen reduction by the vanadiumnitrogenase from Azotobacter chroococcum. Biochem. J., 277, 465–468.Google Scholar
  17. Dilworth, M. J., Eady, R. R., and Eldridge, M. E. (1988). The vanadium nitrogenase of Azotobacter chroococcum. Reduction of acetylene and ethylene to ethane. Biochem. J., 249, 745–751.Google Scholar
  18. Dilworth, M. J., Eldridge, M. E., and Eady, R. R. (1992). Correction for creatine interference with the direct indophenol measurement of ammonia in steady-state nitrogenase assays. Anal. Biochem., 207, 6–10.CrossRefGoogle Scholar
  19. Dilworth, M. J., Eldridge, M. E., and Eady, R. R. (1993). The molybdenum and vanadium nitrogenases of Azotobacter chroococcum: Effect of elevated temperature on nitrogen reduction. Biochem. J., 289, 395–400.Google Scholar
  20. Dilworth, M. J., and Fisher, K. (1998). Elimination of creatine interference with the indophenol measurement of NH3 produced during nitrogenase assays. Anal. Biochem., 256, 242–244.CrossRefGoogle Scholar
  21. Dilworth, M. J., Fisher, K., Kim, C.-H., and Newton, W.E. (1998). Effects on substrate reduction of substitution of histidine-195 by glutamine in the cc-subunit of the MoFe protein of Azotobacter vinelandii nitrogenase. Biochemistry, 37, 17495–17505.CrossRefGoogle Scholar
  22. Dilworth, M. J., and Thorneley, R. N. F. (1981). Nitrogenase of Klebsiella pneumoniae. Hydrazine is a product of azide reduction. Biochem. J., 193, 971–983.Google Scholar
  23. Dubin, P. T. (1960). Assay and characterization of amines by means of 2,4-dinitrofluorobenzene. J. Biol. Chem., 235, 783–786.Google Scholar
  24. Ennor, A. H. (1957). Determination and preparation of N-phosphates of biological origin. Methods Enzymol., 3, 850–856.CrossRefGoogle Scholar
  25. Ensign, S. A. (1995). Reaction of carbon monoxide dehydrogenase from Rhodospirillum rubrum with carbon dioxide, carbonyl sulfide and carbon disulfide. Biochemistry, 34, 5372–5381.CrossRefGoogle Scholar
  26. Erickson, J. A., Nyborg, A. C, Johnson, J. L., Truscott S. M., Gunn, A., Nordmeyer, F. R., and Watt, G. D. (1999). Enhanced efficiency of ATP hydrolysis during nitrogenase catalysis utilizing reductants that form the all-ferrous redox state of the Fe protein. Biochemistry, 38, 14279–14285.CrossRefGoogle Scholar
  27. Fawcett, J. K., and Scott, J. E. (1960). A rapid and precise method for the determination of urea. Clin. Pathol., 13, 156–159.CrossRefGoogle Scholar
  28. Fisher, K., Dilworth, M. J., Kim, C.-H., and Newton, W. E. (2000a). 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, 2970–2979.CrossRefGoogle Scholar
  29. Fisher, K., Dilworth, M. J., Kim, C.-H., and Newton, W. E. (2000/?). Azotobacter vinelandii nitrogenases with substitutions in the FeMo-cofactor environment of the MoFe protein: Effects of acetylene or ethylene on interactions with H+, HCN, and CN. Biochemistry,39, 10855–10865.CrossRefGoogle Scholar
  30. Fisher, K., Dilworth, M. J., and Newton, W. E. (2000c). Differential effects on N2 binding and reduction, HD formation, and azide reduction with a-195Hls- and a-191Gln-substituted MoFe proteins of Azotobacter vinelandii nitrogenase. Biochemistry, 39, 15570–15577.CrossRefGoogle Scholar
  31. Fiske, C. H., and Subbarow, Y. (1925). The colorimetric determination of phosphorus. J. Biol. Chem., 66, 375–400.Google Scholar
  32. Fuchsman, W. H., and Hardy, R. W. F. (1972). Nitrogenase-catalyzed acrylonitrile reductions. Bioinorg. Chem., 7, 195–213.CrossRefGoogle Scholar
  33. Gemoets, J. P., Bravo, M., McKenna, C. E., Leigh, G. J., and Smith, B. E. (1989). Reduction of cyclopropene by NifV" and wild-type nitrogenases from Klebsiella pneumoniae. Biochem. J., 258, 487–491.Google Scholar
  34. Guth, J. H., and Burris, R. H. (1983). Inhibition of nitrogenase-catalyzed ammonia formation by hydrogen. Biochemistry, 22, 5111–5122.CrossRefGoogle Scholar
  35. Hardy, R. W. F., Holsten, R. D., Jackson, E. K., and Burns, R. C. (1968). The acetylene-ethylene assay for N2 fixation: Laboratory and field evaluation. Plant Physiol.,43, 1185–1207.CrossRefGoogle Scholar
  36. Hardy, R. W. F., and Knight, E. (1967). ATP-dependent reduction of azide and hydrogen cyanide by nitrogen-fixing enzymes of Azotobacter vinelandii and Clostridium pasteurianum.Biochim. Biophys. Acta, 139, 69–90.CrossRefGoogle Scholar
  37. Hargis, L. G. (1978). Determination of carbon. In D. F. Boltz and J. A. Howell (Eds.), Colorimetric Determination of Non-Metals. New York: Wiley Publishing.Google Scholar
  38. Jensen, B. B., and Burris, R. H. (1985). Effect of high pN2 and high pD2 on ammonia production, hydrogen evolution, and hydrogen deuteride formation by nitrogenases. Biochemistry, 24, 1141–1147.CrossRefGoogle Scholar
  39. Jensen, B. B., and Burris, R. H. (1986). Nitrous oxide as a substrate and as a competitive inhibitor of nitrogenase. Biochemistry, 25, 1083–1088.CrossRefGoogle Scholar
  40. Johnson, J. L., Tolley, A. M., Erickson, J. A., and Watt, G. D. (1996). Steady-state kinetics studies of dithionite utilization, component protein interaction, and the formation of an oxidized iron protein intermediate during Azotobacter vinelandii nitrogenase catalysis. Biochemistry, 35, 11336–11342.CrossRefGoogle Scholar
  41. Kelly, M. (1968). The kinetics of the reduction of isocyanides, acetylenes and the cyanide ion by nitrogenase preparations from Azotobacter chroococcum and the effects of inhibitors. Biochem. J., 107, 1–6.Google Scholar
  42. Kelly, M., Postgate, J. R., and Richards, R. L. (1967). Reduction of cyanide and isocyanide by nitrogenase of Azotobacter chroococcum. Biochem. J., 102, 1c–3c.Google Scholar
  43. Kumar, M., Lu, W. P., and Ragsdale, S. W. (1994). Binding of carbon disulfide to the site of acetyl-CoA synthesis by the nickel-iron-sulfur protein, carbon monoxide dehydrogenase, from Clostridium thermoaceticum. Biochemistry, 33, 9769–9777.CrossRefGoogle Scholar
  44. Li, J., Burgess, B. K., and Corbin, J. L. (1982). Nitrogenase reactivity: Cyanide as substrate and inhibitor. Biochemistry, 21, 4393–4402.CrossRefGoogle Scholar
  45. Li, J. L., and Burris, R. H. (1983). Influence of pN2 and pD2 on HD formation by various nitrogenases. Biochemistry, 22, 4472–80.CrossRefGoogle Scholar
  46. Liang, J., and Burris, R. H. (1988). Interactions among nitrogen, nitrous oxide and acetylene as substrates and inhibitors of nitrogenase from Azotobacter vinelandii. Biochemistry, 27, 6726–6732.CrossRefGoogle Scholar
  47. Liang, J., and Burris, R. H. (1989). Nitrous oxide reduction and HD formation by nitrogenase from a nijV mutant of Klebsiella pneumoniae. J. Bacteriol, 777,3176–3180.Google Scholar
  48. Lin, J.-K., and Lai, C.-C. (1980). High performance liquid chromatographic determination of naturally occurring primary and secondary amines with dabsyl chloride. Anal. Chem., 52, 630–635.CrossRefGoogle Scholar
  49. Lin-Vien, D., Fateley, W. G., and Davis, L. C. (1989). Estimation of nitrogenase activity in the presence of ethylene biosynthesis by use of deuterated acetylene as a substrate. Appl. Environ. Microbiol., 55, 354–359.Google Scholar
  50. Ljones, T., and Burris, R. H. (1972). Continuous spectrophotometric assay for nitrogenase. Anal. Biochem., 45, 448–452.CrossRefGoogle Scholar
  51. Lockshin, A., and Burris, R. H. (1965). Inhibitors of nitrogen fixation in extracts from Clostridium pasteurianum. Biochim. Biophys. Acta, 111, 1–10.CrossRefGoogle Scholar
  52. Lowe, D. J., Ashby, G. A., Brune, M., Knights, H., Webb, M. R., and Thorneley, R. N. F. (1995). ATP hydrolysis and energy transduction by nitrogenase. In A.I. Tikhonovich, N. A. Provorov, V. I. Romanov and W. E. Newton (Eds.), Nitrogen Fixation: Fundamentals and Applications(pp. 103–108). Dordrecht: Kluwer Academic Publishers.CrossRefGoogle Scholar
  53. Maryan, P. S., and Vorley, W. T. (1979). An improved spectrophotometric method for the determination of ammonia with particular relevance to in vitro nitrogenase activity. Lab. Practice, 28, 251–252.Google Scholar
  54. Mayer, S. M., Niehaus, W. G., and Dean, D. R. (2002). Reduction of short chain alkynes by a nitrogenase a-70Ala-substituted MoFe protein. J. Chem. Soc., Dalton Trans., 802–807.Google Scholar
  55. McKenna, C. E., McKenna, M. C, and Higa, M. T. (1976). Chemical probes of nitrogenase. I. Cyclopropene. Nitrogenase-catalyzed reduction to propene and cyclopropane. J. Amer. Chem. Soc., 98, 4657–4659.CrossRefGoogle Scholar
  56. McKenna, C. E., McKenna, M. C, and Huang, C. W. (1979). Low stereoselectivity in methyl acetylene and cyclopropene reductions by nitrogenase. Proc. Natl. Acad. Sci. U.S.A., 76, 4773–4777.CrossRefGoogle Scholar
  57. Miller, R. W., and Eady, R. R. (1988). Cyanamide: A new substrate for nitrogenase. Biochim. Biophys. Acta, 952, 290–296.CrossRefGoogle Scholar
  58. Nash, T. (1953). The colorimetric estimation of formaldehyde by means of the Hantzsch reaction. Biochem.J., 55,416–421.Google Scholar
  59. O’Donovan, D. J. (1971). Inhibition of the indophenol reaction in the spectrophotometric determination of ammonia. Clin. Chim. Acta, 32, 59–61.CrossRefGoogle Scholar
  60. Ottolenghi, P. (1975). The reversible delipidation of a solubilized sodium-plus-potassiumion-dependent adenosine triphosphatase from the salt gland of the spiny dogfish. Biochem. J., 151, 61–66.Google Scholar
  61. Rasche, M. E., and Seefeldt, L. C. (1997). Reduction of thiocyanate, cyanate, and carbon disulfide by nitrogenase: Kinetic characterization and EPR spectroscopic analysis. Biochemistry, 36, 8574–8585.CrossRefGoogle Scholar
  62. Rubinson, J. F., Burgess, B. K., Corbin, J. L., and Dilworth, M. J. (1985). Nitrogenase reactivity: Azide reduction. Biochemistry, 24, 273–283.CrossRefGoogle Scholar
  63. Rubinson, J. F., Corbin, J. L., and Burgess, B. K. (1983). Nitrogenase reactivity: Methyl isocyanide as substrate and inhibitor. Biochemistry, 22, 6260–6268.CrossRefGoogle Scholar
  64. Schollhorn, R., and Burris, R. H. (1967). Reduction of azide by the N2-fixing enzyme system. Proc. Natl. Acad. Sci. U.S.A., 57, 1317–1323.CrossRefGoogle Scholar
  65. Scott, D. J., Dean, D. R., and Newton, W. E. (1992). Nitrogenase-catalyzed ethane production and CO- sensitive hydrogen evolution from MoFe proteins having amino acid substitutions in an a-subunit FeMo cofactor binding domain. J. Biol. Chem., 267, 20002–20010.Google Scholar
  66. Seefeldt, L. C, and Ensign, S. A. (1994). A continuous, spectrophotometric assay for nitrogenase using the reductant titanium(III) citrate. Anal. Biochem., 221, 379–386.CrossRefGoogle Scholar
  67. Seefeldt, L. C, Rasche, M. E., and Ensign, S. A. (1995). Carbonyl sulfide and carbon dioxide as new substrates, and carbon disulfide as a new inhibitor, of nitrogenase. Biochemistry, 34, 5382–5389.CrossRefGoogle Scholar
  68. Shah, V. K., Davis, L. C, and Brill, W. J. (1972). I. Repression and derepression of the iron-molybdenum and iron proteins of nitrogenase in Azotobacter vinelandii. Biochim. Biophys. Acta, 256,498–511.CrossRefGoogle Scholar
  69. Spies, J. R. (1957). Colorimetric methods for amino acids. Methods Enzymol., 3, 461–471.Google Scholar
  70. Taussky, H. H., Shorr, E., and Kurzmann, G. (1953). A microcolorimetric method for the determination of inorganic phosphorus. J. Biol. Chem., 202, 675–685.Google Scholar
  71. Thorneley, R. N. F., Ashby, G. A., Julius, C, Hunter, J. L., and Webb, M. R. (1991). Nitrogenase of Klebsiella pneumoniae. Reversibility of the reductant-independent magnesium-ATP-cleavage reaction is shown by magnesium-ADP-catalyzed phosphate/water oxygen exchange. Biochem. J., 277,135–141.Google Scholar
  72. Thorneley, R. N. F., and Lowe, D. J. (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, 887–894.Google Scholar
  73. Vaughn, S. A., and Burgess, B. K. (1989). Nitrite, a new substrate for nitrogenase. Biochemistry, 28, 419–424.CrossRefGoogle Scholar
  74. Watt, G. D., and Burns, A. (1977). Kinetics of dithionite ion utilization and ATP hydrolysis for reactions catalyzed by the nitrogenase complex from Azotobacter vinelandii. Biochemistry, 16, 264–270.CrossRefGoogle Scholar
  75. Wherland, S., Burgess, B. K., Stiefel, E. I., and Newton, W. E. (1981). Nitrogenase reactivity: Effects of component ratio on electron flow and distribution during nitrogen fixation. Biochemistry, 20, 5132–5140.CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media Dordrecht 2004

Authors and Affiliations

  • M. J. Dilworth
    • 1
  1. 1.Center for Rhizobium Studies, School of Biological Sciences and BiotechnologyMurdoch UniversityMurdochWestern Australia

Personalised recommendations