Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Review Article
  • Published:

Reversing nitrogen fixation

Nature Reviews Chemistry volume 2, pages 278–289 (2018)Cite this article

Subjects

Abstract

The nitrogen cycle is one of the most important biogeochemical cycles on Earth because nitrogen is an essential nutrient for all life forms. To supplement natural nitrogen fixation, farmers add large amounts of nitrogen-containing fertilizer to their soils such that nitrogen never becomes a limiting nutrient for plant growth. However, of the nitrogen added to fields — most of which is in the form of NH3 and NO3− — only 30–50% is taken up by plants, while the remainder is metabolized by soil microorganisms in processes with detrimental environmental impacts. The first of these processes, that is, nitrification, refers to the biological oxidation of NH3 to NO2− and NO3−, which have low retention in soil and pollute waterways, leading to downstream eutrophication and ultimately ‘dead zones’ (low oxygen zones) in coastal waters, for example, the Gulf of Mexico. In a second process, namely, denitrification, NO3− and NO2− undergo stepwise reduction to N2O and N2. Substantial amounts of the N2O produced in this process escape into the atmosphere, contributing to climate change and ozone destruction. Recent results suggest that nitrification also affords N2O. This Review describes the enzymes involved in NH3 oxidation and N2O production and degradation in the nitrogen cycle. We pay particular attention to the active site structures, the associated coordination chemistry that enables the chemical transformations and the reaction mechanisms.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Fig. 1: The main species and interconversions in the nitrogen cycle.
Fig. 2: Dissimilatory denitrification involves stepwise reduction of NO3− to N2.
Fig. 3: The active site of hydroxylamine oxidoreductase from Nitrosomonas europaea.
Fig. 4: Cytochrome P450 nitric oxide reductase reductively couples NO at a {haem-thiolate} active site.
Fig. 5: Comparison of the bimetallic active site of bacterial NO reductase and an engineered myoglobin.
Fig. 6: Mechanistic proposals for NO reductive coupling catalysed by bacterial NO reductase.
Fig. 7: Bonding in a small molecule Viii–N2O complex.
Fig. 8: Nitrous oxide reductase and its synthetic active site models.

Similar content being viewed by others

References

  1. Lehnert, N., Coruzzi, G., Hegg, E., Seefeldt, L. & Stein, L. NSF Workshop Report: Feeding the World in the 21st Century: Grand Challenges in the Nitrogen Cycle (National Science Foundation: Arlington, VA, 2016).

    Google Scholar 

  2. Canfield, D. E., Glazer, A. N. & Falkowski, P. G. The evolution and future of earth’s nitrogen cycle. Science 330, 192–196 (2010).

    CAS  PubMed  Google Scholar 

  3. Galloway, J. N. et al. Transformation of the nitrogen cycle: recent trends, questions, and potential solutions. Science 320, 889–892 (2008).

    CAS  Google Scholar 

  4. Fields, S. Global nitrogen: cycling out of control. Environ. Health Perspect. 112, A556–A563 (2004).

    PubMed  PubMed Central  Google Scholar 

  5. Smil, V. Nitrogen cycle and world food production. World Agric. 2, 9–13 (2011).

    Google Scholar 

  6. Smil, V. Enriching the Earth: Fritz Haber, Carl Bosch and the Transformation of World Food Production (MIT Press, Cambridge, MA, 2001).

    Google Scholar 

  7. Burgess, B. K. & Lowe, D. J. Mechanism of molybdenum nitrogenase. Chem. Rev. 96, 2983–3012 (1996).

    CAS  PubMed  Google Scholar 

  8. Hoffman, B. M., Lukoyanov, D., Yang, Y.-Z., Dean, D. R. & Seefeldt, L. C. Mechanism of nitrogen fixation by nitrogenase: the next stage. Chem. Rev. 114, 4041–4062 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  9. Burford, R. J. & Fryzuk, M. D. Examining the relationship between coordination mode and reactivity of dinitrogen. Nat. Rev. Chem. 1, 0026 (2017).

    CAS  Google Scholar 

  10. Erisman, J. W., Sutton, M. A., Galloway, J., Klimont, Z. & Winiwarter, W. How a century of ammonia synthesis changed the world. Nat. Geosci. 1, 636–639 (2008).

    CAS  Google Scholar 

  11. Godfray, H. C. J. et al. Food security: the challenge of feeding 9 billion people. Science 327, 812–818 (2010).

    CAS  Google Scholar 

  12. Ferguson, S. J. Nitrogen cycle enzymology. Curr. Opin. Chem. Biol. 2, 182–193 (1998).

    CAS  PubMed  Google Scholar 

  13. Richardson, D. J. & Watmough, N. J. Inorganic nitrogen metabolism in bacteria. Curr. Opin. Chem. Biol. 3, 207–219 (1999).

    CAS  PubMed  Google Scholar 

  14. Maia, L. B. & Moura, J. J. G. How biology handles nitrite. Chem. Rev. 114, 5273–5357 (2014).

    CAS  PubMed  Google Scholar 

  15. Kuypers, M. M. M., Marchant, H. K. & Kartal, B. The microbial nitrogen-cycling network. Nat. Rev. Microbiol. 16, 263–276 (2018).

    CAS  PubMed  Google Scholar 

  16. Ward, B. B. in Nitrification (eds Ward, B. B., Arp, D. J. & Klotz, M.) 3–8 (ASM Press, Sterling, VA, 2011).

  17. Stein, L. Y. & Klotz, M. G. Primer: the nitrogen cycle. Curr. Biol. 26, R94–R98 (2016).

    CAS  PubMed  Google Scholar 

  18. Galloway, J. N., Leach, A. M., Bleeker, A. & Erisman, J. W. A chronology of human understanding of the nitrogen cycle. Phil. Trans. R. Soc. B 368, 20130120 (2013).

    PubMed  Google Scholar 

  19. Bourzac, K. Cleaning up nitrogen on farm lands. Chem. Eng. News 95, 16–19 (2017).

    Google Scholar 

  20. Hooper, A. B., Arciero, D., Bergmann, D. & Hendrich, M. P. in Respiration in Archaea and Bacteria (ed. Zannoni, D.) 121–147 (Springer, Dordrecht, 2005).

  21. Hooper, A. B. & Terry, K. R. Hydroxylamine oxidoreductase of nitrosomonas: production of nitric oxide from hydroxylamine. Biochim. Biophys. Acta. 571, 12–20 (1979).

    CAS  PubMed  Google Scholar 

  22. van de Graaf, A. A. et al. Anaerobic oxidation of ammonium is a biologically mediated process. Appl. Environ. Microbiol. 61, 1246–1251 (1995).

    PubMed  PubMed Central  Google Scholar 

  23. Kartal, B., Geerts, W. & Jetten, M. S. M. Cultivation, detection, and ecophysiology of anaerobic ammonium-oxidizing bacteria. Methods Enzymol. 486, 89–108 (2011).

    CAS  PubMed  Google Scholar 

  24. Kartal, B. et al. Molecular mechanism of anaerobic ammonium oxidation. Nature 479, 127–130 (2011).

    CAS  PubMed  Google Scholar 

  25. Kartal, B. & Keltjens, J. T. Anammox biochemistry: a tale of heme c proteins. Trends Biochem. Sci. 41, 998–1011 (2016).

    CAS  PubMed  Google Scholar 

  26. van de Vossenberg, J. et al. The metagenome of the marine anammox bacterium ‘Candidatus Scalindua profunda’ illustrates the versatility of this globally important nitrogen cycle bacterium. Environ. Microbiol. 15, 1275–1289 (2013).

    PubMed  PubMed Central  Google Scholar 

  27. Daisuke, H. et al. Anammox organism KSU-1 expresses a NirK-type copper-containing nitrite reductase instead of a NirS-type with cytochrome cd 1. FEBS Lett. 586, 1658–1663 (2012).

    Google Scholar 

  28. Dietl, A. et al. The inner workings of the hydrazine synthase multiprotein complex. Nature 527, 394–397 (2015).

    CAS  PubMed  Google Scholar 

  29. Maalcke, W. J. et al. Characterization of anammox hydrazine dehydrogenase, a key N2-producing enzyme in the global nitrogen cycle. J. Biol. Chem. https://doi.org/10.1074/jbc.M116.735530 (2016).

    Article  PubMed  PubMed Central  Google Scholar 

  30. Lam, P. & Kuypers, M. M. M. Microbial nitrogen cycling processes in oxygen minimum Zones. Annu. Rev. Mar. Sci. 3, 317–345 (2011).

    Google Scholar 

  31. Hu, Z. et al. Nitrogen removal by a nitritation-anammox bioreactor at low temperature. Appl. Env. Microbiol. 79, 2807–2812 (2013).

    CAS  Google Scholar 

  32. Zumft, W. G. Cell biology and molecular basis of denitrification. Microbiol. Mol. Biol. Rev. 61, 533–616 (1997).

    CAS  PubMed  PubMed Central  Google Scholar 

  33. Moura, I. & Moura, J. J. G. Structural aspects of denitrifying enzymes. Curr. Opin. Chem. Biol. 5, 168–175 (2001).

    CAS  PubMed  Google Scholar 

  34. US Environmental Protection Agency. Overview of greenhouse gases. EPA.gov https://www.epa.gov/ghgemissions/overview-greenhouse-gases (2016).

  35. Kandeler, E., Poll, C., Frankenberger Jr, W. T. & Tabatabai, M. A. in Methods in Soil Enzymology (ed. Dick, R. P.) (Soil Science Society of America, Madison, 2011).

  36. Hodge, A., Robinson, D. & Fitter, A. Are microorganisms more effective than plants at competing for nitrogen? Trends Plant Sci. 5, 304–308 (2000).

    CAS  PubMed  Google Scholar 

  37. Masclaux-Daubresse, C. et al. Nitrogen uptake, assimilation and remobilization in plants: challenges for sustainable and productive agriculture. Ann. Bot. 105, 1141–1157 (2010).

    PubMed  PubMed Central  Google Scholar 

  38. Chain, P. et al. Complete genome sequence of the ammonia-oxidizing bacterium and obligate chemolithoautotroph Nitrosomonas europaea. J. Bacteriol. 185, 2759–2773 (2003).

    CAS  PubMed  Google Scholar 

  39. Heil, J., Vereecken, H. & Brüggemann, N. A review of chemical reactions of nitrification intermediates and their role in nitrogen cycling and nitrogen trace gas formation in soil. Eur. J. Soil Sci. 67, 23–39 (2016).

    CAS  Google Scholar 

  40. Gilch, S., Meyer, O. & Schmidt, I. A soluble form of ammonia monooxygenase in Nitrosomonas europaea. Biol. Chem. 390, 863–873 (2009).

    CAS  PubMed  Google Scholar 

  41. Robertson, G. P. & Groffmann, P. M. in Soil Microbiology, Ecology, and Biochemistry (ed. Eldor, P.) 341–364 (Elsevier, Amsterdam, 2007).

  42. Zahn, J. A. & DiSpirito, A. A. Membrane-associated methane monooxygenase from Methylococcus capsulatus (Bath). J. Bacteriol. 178, 1018–1029 (1996).

    CAS  PubMed  PubMed Central  Google Scholar 

  43. Hooper, A. B. & Terry, K. R. Specific inhibitors of ammonia oxidation in Nitrosomonas. J. Bacteriol. 115, 480–485 (1973).

    CAS  PubMed  Google Scholar 

  44. Ensign, S. A., Hyman, M. R. & Arp, D. J. In vitro activation of ammonia monooxygenase from Nitrosomonas europaea by copper. J. Bacteriol. 175, 1971–1980 (1993).

    CAS  PubMed  PubMed Central  Google Scholar 

  45. Zahn, J. A., Arciero, D. M., Hooper, A. B. & DiSpirito, A. A. Evidence for an iron center in the ammonia monooxygenase from Nitrosomonas europaea. FEBS Lett. 397, 35–38 (1996).

    CAS  PubMed  Google Scholar 

  46. Hooper, A. B., Terry, K. R. & Maxwell, P. C. Hydroxylamine oxidoreductase of Nitrosomonas. Oxidation of diethyldithiocarbamate concomitant with stimulation of nitrite synthesis. Biochim. Biophys. Acta, Bioenerg. 462, 141–152 (1977).

    CAS  Google Scholar 

  47. Ramaiah, A. & Nicholas, D. J. D. The synthesis of ATP and the incorporation of 32P by cell-free preparations from Nitrosomonas europaea. Biochim. Biophys. Acta, Gen. Subj. 86, 459–465 (1964).

    CAS  Google Scholar 

  48. Igarashi, N., Moriyama, H., Fujiwara, T., Fukumori, Y. & Tanaka, N. The 2.8 Å structure of hydroxylamine oxidoreductase from a nitrifying chemoautotrophic bacterium. Nitrosomonas europaea. Nat. Struct. Biol. 4, 276 (1997).

    CAS  PubMed  Google Scholar 

  49. Cedervall, P., Hooper, A. B. & Wilmot, C. M. Structural studies of hydroxylamine oxidoreductase reveal a unique heme cofactor and a previously unidentified interaction partner. Biochemistry 52, 6211–6218 (2013).

    CAS  PubMed  Google Scholar 

  50. Collins, M. J., Arciero, D. M. & Hooper, A. B. Optical spectropotentiometric resolution of the hemes of hydroxylamine oxidoreductase. Heme quantitation and pH dependence of E m*. J. Biol. Chem. 268, 14655–14662 (1993).

    CAS  PubMed  Google Scholar 

  51. Arciero, D. M., Golombek, A., Hendrich, M. P. & Hooper, A. B. Correlation of optical and EPR signals with the P460 heme of hydroxylamine oxidoreductase from Nitrosomonas europaea. Biochemistry 37, 523–529 (1998).

    CAS  PubMed  Google Scholar 

  52. Caranto, J. D. & Lancaster, K. M. Nitric oxide is an obligate bacterial nitrification intermediate produced by hydroxylamine oxidoreductase. Proc. Natl Acad. Sci. USA 114, 8217–8222 (2017).

    CAS  PubMed  Google Scholar 

  53. Cabail, M. Z. & Pacheco, A. A. Selective one-electron reduction of Nitrosomonas europaea hydroxylamine oxidoreductase with nitric oxide. Inorg. Chem. 42, 270–272 (2003).

    CAS  PubMed  Google Scholar 

  54. Fernández, M. L., Estrin, D. A. & Bari, S. E. Theoretical insight into the hydroxylamine oxidoreductase mechanism. J. Inorg. Biochem. 102, 1523–1530 (2008).

    PubMed  Google Scholar 

  55. Arciero, D. M., Balny, C. & Hooper, A. B. Spectroscopic and rapid kinetic studies of reduction of cytochrome c554 by hydroxylamine oxidoreductase from Nitrosomonas europaea. Biochemistry 30, 11466–11472 (1991).

    CAS  PubMed  Google Scholar 

  56. Walker, F. A. Nitric oxide interaction with insect nitrophorins and thoughts on the electron configuration of the {FeNO}6 complex. J. Inorg. Biochem. 99, 216–236 (2005).

    CAS  PubMed  Google Scholar 

  57. Maalcke, W. J. et al. Structural basis of biological NO generation by octaheme oxidoreductases. J. Biol. Chem. 289, 1228–1242 (2014).

    CAS  PubMed  Google Scholar 

  58. Pearson, A. R. et al. The crystal structure of cytochrome P460 of Nitrosomonas europaea reveals a novel cytochrome fold and heme−protein cross-link. Biochemistry 46, 8340–8349 (2007).

    CAS  PubMed  PubMed Central  Google Scholar 

  59. Andersson, K. K., Kent, T. A., Lipscomb, J. D., Hooper, A. B. & Münck, E. Mössbauer, EPR, and optical studies of the P-460 center of hydroxylamine oxidoreductase from Nitrosomonas. A ferrous heme with an unusually large quadrupole splitting. J. Biol. Chem. 259, 6833–6840 (1984).

    CAS  PubMed  Google Scholar 

  60. Erickson, R. H. & Hooper, A. B. Preliminary characterization of a variant co-binding heme protein from Nitrosomonas. Biochim. Biophys. Acta, Bioenerg. 275, 231–244 (1972).

    CAS  Google Scholar 

  61. Miller, D. J., Wood, P. M. & Nicholas, D. J. D. Further characterization of cytochrome P-460 in Nitrosomonas europaea. Microbiology 130, 3049–3054 (1984).

    CAS  Google Scholar 

  62. Caranto, J. D., Vilbert, A. C. & Lancaster, K. M. Nitrosomonas europaea cytochrome P460 is a direct link between nitrification and nitrous oxide emission. Proc. Natl Acad. Sci. USA 113, 14704–14709 (2016).

    CAS  PubMed  Google Scholar 

  63. Vilbert, A. C., Caranto, J. D. & Lancaster, K. Influences of the heme–lysine crosslink in cytochrome P460 over redox catalysis and nitric oxide sensitivity. Chem. Sci. 9, 368–379 (2018).

    CAS  PubMed  Google Scholar 

  64. Goodrich, L. E. et al. Electronic structure and biologically relevant reactivity of low-spin {FeNO}8 porphyrin model complexes: new insight from a bis-picket fence porphyrin. Inorg. Chem. 52, 7766–7780 (2013).

    CAS  PubMed  Google Scholar 

  65. Berto, T. C., Praneeth, V. K. K., Goodrich, L. E. & Lehnert, N. Iron–porphyrin NO complexes with covalently attached N-donor ligands: formation of a stable six-coordinate species in solution. J. Am. Chem. Soc. 131, 17116–17126 (2009).

    CAS  PubMed  Google Scholar 

  66. Lehnert, N. et al. Oriented single-crystal nuclear resonance vibrational spectroscopy of [Fe(TPP)(MI)(NO)]: quantitative assessment of the trans effect of NO. Inorg. Chem. 49, 7197–7215 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  67. Hunt, A. P. & Lehnert, N. Heme–nitrosyls: electronic structure implications for function in biology. Acc. Chem. Res. 48, 2117–2125 (2015).

    CAS  PubMed  Google Scholar 

  68. Herzik Jr., M. A., Jonnalagadda, R., Kuriyan, J. & Marletta, M. A. Structural insights into the role of iron–histidine bond cleavage in nitric oxide-induced activation of H-NOX gas sensor proteins. Proc. Natl Acad. Sci. USA 111, E4156–E4164 (2014).

    CAS  PubMed  Google Scholar 

  69. Plate, L. & Marletta, M. A. Nitric oxide-sensing H-NOX proteins govern bacterial communal behavior. Trends Biochem. Sci. 38, 566–575 (2013).

    CAS  PubMed  Google Scholar 

  70. Zahn, J. A., Duncan, C. & DiSpirito, A. A. Oxidation of hydroxylamine by cytochrome P-460 of the obligate methylotroph Methylococcus capsulatus Bath. J. Bacteriol. 176, 5879–5887 (1994).

    CAS  PubMed  PubMed Central  Google Scholar 

  71. Luesken, F. A. et al. Simultaneous nitrite-dependent anaerobic methane and ammonium oxidation processes. Appl. Environ. Microbiol. 77, 6802–6807 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  72. Beaumont, H. J. E., Lens, S. I., Westerhoff, H. V. & van Spanning, R. J. M. Novel nirK cluster genes in Nitrosomonas europaea are required for NirK-dependent tolerance to nitrite. J. Bacteriol. 187, 6849–6851 (2005).

    CAS  PubMed  PubMed Central  Google Scholar 

  73. Beaumont, H. J. E., van Schooten, B., Lens, S. I., Westerhoff, H. V. & van Spanning, R. J. M. Nitrosomonas europaea expresses a nitric oxide reductase during nitrification. J. Bacteriol. 186, 4417–4421 (2004).

    CAS  PubMed  PubMed Central  Google Scholar 

  74. Wrage, N., Velthof, G. L., van Beusichem, M. L. & Oenema, O. Role of nitrifier denitrification in the production of nitrous oxide. Soil Biol. Biochem. 33, 1723–1732 (2001).

    CAS  Google Scholar 

  75. Meincke, M., Bock, E., Kastrau, D. & Kroneck, P. M. H. Nitrite oxidoreductase from Nitrobacter hamburgensis: redox centers and their catalytic role. Arch. Microbiol. 158, 127–131 (1992).

    Google Scholar 

  76. Daims, H., Lücker, S. & Wagner, M. A new perspective on microbes formerly known as nitrite-oxidizing bacteria. Trends Microbiol. 24, 699–712 (2016).

    CAS  PubMed  Google Scholar 

  77. Lehnert, N., Berto, T. C., Galinato, M. G. I. & Goodrich, L. E. in The Handbook of Porphyrin Science (eds Kadish, K. M., Smith, K. M. & Guilard, R.) 1–247 (World Scientific, New Jersey, 2011).

  78. Ravishankara, A. R., Daniel, J. S. & Portmann, R. W. Nitrous oxide (N2O): the dominant ozone-depleting substance emitted in the 21st century. Science 326, 123–125 (2009).

    CAS  Google Scholar 

  79. Torres, M. J. et al. Nitrous oxide metabolism in nitrate-reducing bacteria: physiology and regulatory mechanisms. Advances in Microbial Physiology (ed. Poole, R. K.) 38, 353–432 (Elsevier, 2016).

    Google Scholar 

  80. Speelman, A. L. & Lehnert, N. Heme versus non-heme iron-nitroxyl {FeN(H)O}8 complexes: electronic structure and biologically relevant reactivity. Acc. Chem. Res. 47, 1106–1116 (2014).

    CAS  PubMed  Google Scholar 

  81. Laughlin, R. J. & Stevens, R. J. Evidence for fungal dominance of denitrification and codenitrification in a grassland soil. Soil Sci. Soc. Am. J. 66, 1540–1548 (2002).

    CAS  Google Scholar 

  82. Crenshaw, C. L., Lauber, C., Sinsabaugh, R. L. & Stavely, L. K. Fungal control of nitrous oxide production in semiarid grassland. Biogeochem. 87, 17–27 (2008).

    CAS  Google Scholar 

  83. Long, A., Heitman, J., Tobias, C., Philips, R. & Song, B. Co-occurring anammox, denitrification, and codenitrification in agricultural soils. Appl. Environ. Microbiol. 79, 168–176 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  84. Wankel, S. D. et al. Evidence for fungal and chemodenitrification based N2O flux from nitrogen impacted coastal sediments. Nat. Commun. 8, 15595 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  85. Lewicka-Szczebak, D., Augustin, J., Giesemann, A. & Well, R. Quantifying N2O reduction to N2 based on N2O isotopocules — validation with independent methods (helium incubation and 15N gas flux method). Biogeoscience 14, 711–732 (2017).

    CAS  Google Scholar 

  86. Rohe, L., Well, R. & Lewicka-Szczebak, D. Use of oxygen isotopes to differentiate between nitrous oxide produced by fungi or bacteria during denitrification. Rapid Commun. Mass Spectrom. 31, 1297–1312 (2017).

    CAS  PubMed  Google Scholar 

  87. Caranto, J. D., Weitz, A., Hendrich, M. P. & Kurtz Jr., D. M. The nitric oxide reductase mechanism of a flavo–diiron protein: identification of active-site intermediates and products. J. Am. Chem. Soc. 136, 7981–7992 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  88. Zheng, S. et al. The functional model complex [Fe2(BPMP)(OPr)(NO)2](BPh4)2 provides insight into the mechanism of flavodiiron NO reductases. J. Am. Chem. Soc. 135, 4902–4905 (2013).

    CAS  PubMed  Google Scholar 

  89. Maeda, K. et al. N2O production, a widespread trait in fungi. Sci. Rep. 5, 9697 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  90. Shoun, H. & Tanimoto, T. Denitrification by the fungus Fusarium oxysporum and involvement of cytochrome P-450 in the respiratory nitrite reduction. J. Biol. Chem. 266, 11078–11082 (1991).

    CAS  PubMed  Google Scholar 

  91. Shoun, H., Sudo, Y., Seto, Y. & Beppu, T. Purification and properties of a cyotchrome P-450 of a fungus. Fusarium oxysporum. J. Biochem. 94, 1219–1229 (1983).

    CAS  PubMed  Google Scholar 

  92. Nittler, M. P., Hocking-Murray, D., Foo, C. K. & Sil, A. Identification of Histoplasma capsulatum transcripts induced in response to reactive nitrogen species. Mol. Biol. Cell 16, 4792–4813 (2005).

    CAS  PubMed  PubMed Central  Google Scholar 

  93. Yang, H., Gandhi, H., Ostrom, N. E. & Hegg, E. L. Isotopic fractionation by a fungal P450 nitric oxide reductase during the production of N2O. Environ. Sci. Technol. 48, 10707–10715 (2014).

    CAS  PubMed  Google Scholar 

  94. Usuda, K., Toritsuka, N., Matsuo, Y., Kim, D. H. & Shoun, H. Denitrification by the fungus Cylindrocarpon tonkinense: anaerobic cell growth and two isozyme forms of cytochrome P-450nor. Appl. Environ. Microbiol. 61, 883–889 (1995).

    CAS  PubMed  PubMed Central  Google Scholar 

  95. Tsuruta, S. et al. Denitrification by yeasts and occurrence of cytochrome P450nor in Trichosporon cutaneum. FEMS Microbiol. Lett. 168, 105–110 (1998).

    CAS  PubMed  Google Scholar 

  96. Shoun, H., Kim, D.-H., Uchiyama, H. & Sugiyama, J. Denitrification by fungi. FEMS Microbiol. Lett. 94, 277–281 (1992).

    CAS  Google Scholar 

  97. McQuarters, A. B., Wolf, M. W., Hunt, A. P. & Lehnert, N. 1958–2014: after 56 years of research, cytochrome P450 reactivity finally explained. Angew. Chem. Int. Ed. 53, 4750–4752 (2014).

    CAS  Google Scholar 

  98. McQuarters, A. B., Wirgau, N. E. & Lehnert, N. Model complexes of key intermediates in fungal cytochrome P450 nitric oxide reductase (P450nor). Curr. Opin. Chem. Biol. 19, 82–89 (2014).

    CAS  PubMed  Google Scholar 

  99. Shoun, H., Fushinobu, S., Jiang, L., Kim, S.-W. & Wakagi, T. Fungal denitrification and nitric oxide reductase cytochrome P450nor. Phil. Trans. R. Soc. B 367, 1186–1194 (2012).

    CAS  PubMed  Google Scholar 

  100. Shiro, Y. et al. Spectroscopic and kinetic studies on reaction of cytochrome P450nor with nitric oxide. J. Biol. Chem. 270, 1617–1623 (1995).

    CAS  PubMed  Google Scholar 

  101. Shimizu, H. et al. Proton delivery in NO reduction by fungal nitric-oxide reductase. Cryogenic crystallography, spectroscopy, and kinetics of ferric-NO complexes of wild-type and mutant enzymes. J. Biol. Chem. 275, 4816–4826 (2000).

    CAS  PubMed  Google Scholar 

  102. Obayashi, E. et al. Unique binding of nitric oxide to ferric nitric oxide reductase from Fusarium oxysporum elucidated with infrared, resonance Raman, and X-ray absorption spectroscopies. J. Am. Chem. Soc. 119, 7807–7816 (1997).

    CAS  Google Scholar 

  103. Paulat, F. & Lehnert, N. Electronic structure of ferric heme nitrosyl complexes with thiolate coordination. Inorg. Chem. 46, 1547–1549 (2007).

    CAS  PubMed  Google Scholar 

  104. Praneeth, V. K. K. et al. Electronic structure of six-coordinate iron(iii)–porphyrin NO adducts: the elusive iron(iii)–NO(radical) state and its influence on the properties of these complexes. J. Am. Chem. Soc. 130, 15288–15303 (2008).

    CAS  PubMed  Google Scholar 

  105. Lehnert, N., Scheidt, W. R. & Wolf, M. W. Structure and bonding in heme–nitrosyl complexes and implications for biology. Struct. Bond. 154, 155–223 (2014).

    CAS  Google Scholar 

  106. Soldatova, A. V., Ibrahim, M., Olson, J. S., Czernuszewicz, R. S. & Spiro, T. G. New light on NO Bonding in Fe(iii) heme proteins from resonance Raman spectroscopy and DFT modeling. J. Am. Chem. Soc. 132, 4614–4625 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  107. Enemark, J. H. & Feltham, R. D. Principles of structure, bonding, and reactivity for metal nitrosyl complexes. Coord. Chem. Rev. 13, 339–406 (1974).

    CAS  Google Scholar 

  108. Wang, Y. & Averill, B. A. Direct observation by FTIR spectroscopy of the ferrous heme−NO+ intermediate in reduction of nitrite by a dissimilatory heme cd 1 nitrite reductase. J. Am. Chem. Soc. 118, 3972–3973 (1996).

    CAS  Google Scholar 

  109. Oshima, R. et al. Structural evidence for direct hydride transfer from NADH to cytochrome P450nor. J. Mol. Biol. 342, 207–217 (2004).

    CAS  PubMed  Google Scholar 

  110. Riplinger, C. & Neese, F. The reaction mechanism of cytochrome P450 NO reductase: a detailed quantum mechanics/molecular mechanics study. ChemPhysChem. 12, 3192–3203 (2011).

    CAS  PubMed  Google Scholar 

  111. Krámos, B., Menyhárd, D. K. & Oláh, J. Direct hydride shift mechanism and stereoselectivity of P450nor confirmed by QM/MM calculations. J. Phys. Chem. B 116, 872–885 (2012).

    PubMed  Google Scholar 

  112. Riplinger, C. et al. New insights into the nature of observable reaction intermediates in cytochrome P450 NO reductase by using a combination of spectroscopy and quantum mechanics/molecular mechanics calculations. Chem. Eur. J. 20, 1602–1614 (2014).

    CAS  PubMed  Google Scholar 

  113. Obayashi, E., Takahashi, S. & Shiro, Y. Electronic structure of reaction intermediate of cytochrome P450nor in its nitric oxide reduction. J. Am. Chem. Soc. 120, 12964–12965 (1998).

    CAS  Google Scholar 

  114. Lehnert, N., Praneeth, V. K. K. & Paulat, F. Electronic structure of iron(ii)–porphyrin nitroxyl complexes: molecular mechanism of fungal nitric oxide reductase (P450nor). J. Comput. Chem. 27, 1338–1351 (2006).

    CAS  PubMed  Google Scholar 

  115. Yosca, T. H. et al. Iron(iv) hydroxide pK a and the role of thiolate ligation in C–H bond activation by cytochrome P450. Science 342, 825–829 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  116. Yosca, T. H. et al. Setting an upper limit on the myoglobin iron(iv) hydroxide pK a: insight into axial ligand tuning in heme protein catalysis. J. Am. Chem. Soc. 136, 9124–9131 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  117. Lin, R. & Farmer, P. J. The HNO adduct of myoglobin: synthesis and characterization. J. Am. Chem. Soc. 122, 2393–2394 (2000).

    CAS  Google Scholar 

  118. Lin, Y.-W. Rational design of metalloenzymes: from single to multiple active sites. Coord. Chem. Rev. 336, 1–27 (2017).

    CAS  Google Scholar 

  119. Wasser, I. M., de Vries, S., Moënne-Loccoz, P., Schröder, I. & Karlin, K. D. Nitric oxide in biological denitrification: Fe/Cu metalloenzymes and metal complex NOx redox chemistry. Chem. Rev. 102, 1201–1234 (2002).

    CAS  PubMed  Google Scholar 

  120. Zumft, W. G. Nitric oxide reductases of prokaryotes with emphasis on the respiratory, heme–copper oxidase type. J. Inorg. Biochem. 99, 194–215 (2005).

    CAS  PubMed  Google Scholar 

  121. Saraste, M. & Castresana, J. Cytochrome oxidase evolved by tinkering with denitrification enzymes. FEBS Lett. 341, 1–4 (1994).

    CAS  PubMed  Google Scholar 

  122. Shiro, Y. Structure and function of bacterial nitric oxide reductases. Biochim. Biophys. Acta, Bioenerg. 1817, 1907–1913 (2012).

    CAS  Google Scholar 

  123. Hino, T. et al. Structural basis of biological N2O generation by bacterial nitric oxide reductase. Science 330, 1666–1670 (2010).

    CAS  PubMed  Google Scholar 

  124. Timóteo, C. G. et al. Low-spin heme b 3 in the catalytic center of nitric oxide reductase from Pseudomonas nautica. Biochemistry 50, 4251–4262 (2011).

    PubMed  PubMed Central  Google Scholar 

  125. Blomberg, M. R. How quantum chemistry can solve fundamental problems in bioenergetics. Int. J. Quantum Chem. 115, 1197–1201 (2015).

    CAS  Google Scholar 

  126. Al-Attar, S. & de Vries, S. An electrogenic nitric oxide reductase. FEBS Lett. 589, 2050–2057 (2015).

    CAS  PubMed  Google Scholar 

  127. Gonska, N. et al. Characterization of the quinol-dependent nitric oxide reductase from the pathogen Neisseria meningitidis, an electrogenic enzyme. Sci. Rep. 8, 3637 (2018).

    PubMed  PubMed Central  Google Scholar 

  128. Richardson, D., Felgate, H., Watmough, N., Thomson, A. & Baggs, E. Mitigating release of the potent greenhouse gas N2O from the nitrogen cycle — could enzymic regulation hold the key? Trends Biotechnol. 27, 388–397 (2009).

    CAS  PubMed  Google Scholar 

  129. Kakishima, K., Shiratsuchi, A., Taoka, A., Nakanishi, Y. & Fukumori, Y. Participation of nitric oxide reductase in survival of Pseudomonas aeruginosa in LPS-activated macrophages. Biochem. Biophys. Res. Commun. 355, 587–591 (2007).

    CAS  PubMed  Google Scholar 

  130. Yeung, N. et al. Rational design of a structural and functional nitric oxide reductase. Nature 462, 1079–1082 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  131. Butler, C. S., Seward, H. E., Greenwood, C. & Thomson, A. J. Fast cytochrome bo from Escherichia coli binds two molecules of nitric oxide at CuB. Biochemistry 36, 16259–16266 (1997).

    CAS  PubMed  Google Scholar 

  132. Girsch, P. & de Vries, S. Purification and initial kinetic and spectroscopic characterization of NO reductase from Paracoccus denitrificans. Biochim. Biophys. Acta, Bioenergy 1318, 202–216 (1997).

    CAS  Google Scholar 

  133. Watmough, N. J. et al. The dinuclear center of cytochrome bo 3 from Escherichia coli. J. Bioenerg. Biomembr. 30, 55–62 (1998).

    CAS  PubMed  Google Scholar 

  134. Hendriks, J. et al. The active site of the bacterial nitric oxide reductase is a dinuclear iron center. Biochemistry 37, 13102–13109 (1998).

    CAS  PubMed  Google Scholar 

  135. Moënne-Loccoz, P. & de Vries, S. Structural characterization of the catalytic high-spin heme b of nitric oxide reductase: a resonance Raman study. J. Am. Chem. Soc. 120, 5147–5152 (1998).

    Google Scholar 

  136. Cheesman, M. R., Zumft, W. G. & Thomson, A. J. The MCD and EPR of the heme centers of nitric oxide reductase from Pseudomonas stutzeri: evidence that the enzyme is structurally related to the heme–copper oxidases. Biochemistry 37, 3994–4000 (1998).

    CAS  PubMed  Google Scholar 

  137. Flock, U., Reimann, J. & Ädelroth, P. Proton transfer in bacterial nitric oxide reductase. Biochem. Soc. Trans. 34, 188–190 (2006).

    CAS  PubMed  Google Scholar 

  138. Speelman, A. L. et al. Unusual synthetic pathway for an {Fe(NO)2}9 dinitrosyl iron complex (DNIC) and insight into DNIC electronic structure via nuclear resonance vibrational spectroscopy. Inorg. Chem. 55, 5485–5501 (2016).

    CAS  PubMed  Google Scholar 

  139. Praneeth, V. K. K., Näther, C., Peters, G. & Lehnert, N. Spectroscopic properties and electronic structure of five- and six-coordinate iron(ii) porphyrin NO complexes: effect of the axial N-donor ligand. Inorg. Chem. 45, 2795–2811 (2006).

    CAS  PubMed  Google Scholar 

  140. Berto, T. C. et al. Characterization of the bridged hyponitrite complex {[Fe(OEP)]2(μ-N2O2)}: reactivity of hyponitrite complexes and biological relevance. Inorg. Chem. 53, 6398–6414 (2014).

    CAS  PubMed  Google Scholar 

  141. Blomberg, M. R. A. Can reduction of NO to N2O in cytochrome c dependent nitric Oxide reductase proceed through a trans-mechanism? Biochemistry 56, 120–131 (2017).

    CAS  PubMed  Google Scholar 

  142. Matsumura, H., Hayashi, T., Chakraborty, S., Lu, Y. & Moënne-Loccoz, P. The production of nitrous oxide by the heme/nonheme diiron center of engineered myoglobins (FeBMbs) proceeds through a trans-iron-nitrosyl dimer. J. Am. Chem. Soc. 136, 2420–2431 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  143. Matsumura, H., Chakraborty, S., Reed, J., Lu, Y. & Moënne-Loccoz, P. Effect of outer-sphere side chain substitutions on the fate of the trans iron–nitrosyl dimer in heme/nonheme engineered myoglobins (FeBMbs): insights into the mechanism of denitrifying NO reductases. Biochemistry 55, 2091–2099 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  144. Bhagi-Damodaran, A., Petrik, I. & Lu, Y. Using biosynthetic models of heme-copper oxidase and nitric oxide reductase in myoglobin to elucidate structural features responsible for enzymatic activities. Isr. J. Chem. 56, 773–790 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  145. Chakraborty, S. et al. Recent advances in biosynthetic modeling of nitric oxide reductases and insights gained from nuclear resonance vibrational and other spectroscopic studies. Inorg. Chem. 54, 9317–9329 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  146. Collman, J. P. et al. A functional nitric oxide reductase model. Proc. Natl Acad. Sci. USA 105, 15660–15665 (2008).

    CAS  PubMed  Google Scholar 

  147. Blomberg, M. R. A. & Siegbahn, P. E. M. Mechanism for N2O generation in bacterial nitric oxide reductase: a quantum chemical study. Biochemistry 51, 5173–5186 (2012).

    CAS  PubMed  Google Scholar 

  148. Blomberg, L. M., Blomberg, M. R. A. & Siegbahn, P. E. M. Reduction of nitric oxide in bacterial nitric oxide reductase — a theoretical model study. Biochim. Biophys. Acta, Bioenerg. 1757, 240–252 (2006).

    CAS  Google Scholar 

  149. Ye, R. W., Averill, B. A. & Tiedje, J. M. Denitrification: production and consumption of nitric oxide. Appl. Environ. Microbiol. 60, 1053–1058 (1994).

    CAS  PubMed  PubMed Central  Google Scholar 

  150. Hayashi, T. et al. Spectroscopic characterization of mononitrosyl complexes in heme–nonheme diiron centers within the myoglobin scaffold (FeBMbs): relevance to denitrifying NO reductase. Biochemistry 50, 5939–5947 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  151. Tolman, W. B. Binding and activation of N2O at transition-metal centers: recent mechanistic insights. Angew. Chem. Int. Ed. 49, 1018–1024 (2010).

    CAS  Google Scholar 

  152. Severin, K. Synthetic chemistry with nitrous oxide. Chem. Soc. Rev. 44, 6375–6386 (2015).

    CAS  PubMed  Google Scholar 

  153. Parmon, V. N., Panov, G. I., Uriarte, A. & Noskov, A. S. Nitrous oxide in oxidation chemistry and catalysis: application and production. Catal. Today 100, 115–131 (2005).

    CAS  Google Scholar 

  154. Gorelsky, S. I., Ghosh, S. & Solomon, E. I. Mechanism of N2O reduction by the μ4-S tetranuclear CuZ cluster of nitrous oxide reductase. J. Am. Chem. Soc. 128, 278–290 (2006).

    CAS  PubMed  Google Scholar 

  155. Zeng, R., Feller, M., Ben-David, Y. & Milstein, D. Hydrogenation and hydrosilylation of nitrous oxide homogeneously catalyzed by a metal complex. J. Am. Chem. Soc. 139, 5720–5723 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  156. Leont’ev, A. V., Fomicheva, O. A., Proskurnina, V. M. V. & Zefirov, S. N. S. Modern chemistry of nitrous oxide. Russ. Chem. Rev. 70, 91–104 (2001).

    Google Scholar 

  157. Armor, J. N. & Taube, H. Formation and reactions of [(NH3)5RuN2O2+]. J. Am. Chem. Soc. 91, 6874–6876 (1969).

    CAS  Google Scholar 

  158. Diamantis, A. A. & Sparrow, G. J. Nitrous oxide complexes: the isolation of pentammine(dinitrogen oxide)ruthenium(ii) tetrafluoroborate. Chem. Commun. 819–820 (1970).

  159. Pamplin, C. B., Ma, E. S. F., Safari, N., Rettig, S. J. & James, B. R. The nitrous oxide complex, RuCl2(η1-N2O)(P−N)(PPh3) (P−N = [o-(N,N-dimethylamino)phenyl]diphenylphosphine); low temperature conversion of N2O to N2 and O2. J. Am. Chem. Soc. 123, 8596–8597 (2001).

    CAS  PubMed  Google Scholar 

  160. Paulat, F. et al. Spectroscopic properties and electronic structure of pentammineruthenium(ii) dinitrogen oxide and corresponding nitrosyl complexes: binding mode of N2O and reactivity. Inorg. Chem. 43, 6979–6994 (2004).

    CAS  PubMed  Google Scholar 

  161. Piro, N. A., Lichterman, M. F., Harman, W. H. & Chang, C. J. A structurally characterized nitrous oxide complex of vanadium. J. Am. Chem. Soc. 133, 2108–2111 (2011).

    CAS  PubMed  Google Scholar 

  162. Dell’Acqua, S., Pauleta, S. R., Moura, I. & Moura, J. J. G. The tetranuclear copper active site of nitrous oxide reductase: the CuZ center. J. Biol. Inorg. Chem. 16, 183–194 (2011).

    PubMed  Google Scholar 

  163. Riester, J., Zumft, W. G. & Kroneck, P. M. H. Nitrous oxide reductase from Pseudomonas stutzeri. Eur. J. Biochem. 178, 751–762 (1989).

    CAS  PubMed  Google Scholar 

  164. McEwan, A. G., Greenfield, A. J., Wetzstein, H. G., Jackson, J. B. & Ferguson, S. J. Nitrous oxide reduction by members of the family Rhodospirillaceae and the nitrous oxide reductase of Rhodopseudomonas capsulata. J. Bacteriol. 164, 823–830 (1985).

    CAS  PubMed  PubMed Central  Google Scholar 

  165. Hole, U. H. et al. Characterization of the membranous denitrification enzymes nitrite reductase (cytochrome cd 1) and copper-containing nitrous oxide reductase from Thiobacillus denitrificans. Arch. Microbiol. 165, 55–61 (1996).

    CAS  PubMed  Google Scholar 

  166. Prudêncio, M. et al. Purification, characterization, and preliminary crystallographic study of copper-containing nitrous oxide reductase from Pseudomonas nautica 617. Biochemistry 39, 3899–3907 (2000).

    PubMed  Google Scholar 

  167. Brown, K. et al. A novel type of catalytic copper cluster in nitrous oxide reductase. Nat. Struct. Biol. 7, 191–195 (2000).

    CAS  PubMed  Google Scholar 

  168. Chen, P., Cabrito, I., Moura, J. J. G., Moura, I. & Solomon, E. I. Spectroscopic and electronic structure studies of the μ4-sulfide bridged tetranuclear CuZ cluster in N2O reductase: molecular insight into the catalytic mechanism. J. Am. Chem. Soc. 124, 10497–10507 (2002).

    CAS  PubMed  Google Scholar 

  169. Johnston, E. M. et al. Determination of the active form of the tetranuclear copper sulfur cluster in nitrous oxide reductase. J. Am. Chem. Soc. 136, 614–617 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  170. Pomowski, A., Zumft, W. G., Kroneck, P. M. H. & Einsle, O. N2O binding at a [4Cu:2S] copper–sulphur cluster in nitrous oxide reductase. Nature 477, 234–237 (2011).

    CAS  PubMed  Google Scholar 

  171. Paraskevopoulos, K., Antonyuk, S. V., Sawers, R. G., Eady, R. R. & Hasnain, S. S. Insight into catalysis of nitrous oxide reductase from high-resolution structures of resting and inhibitor-bound enzyme from Achromobacter cycloclastes. J. Mol. Biol. 362, 55–65 (2006).

    CAS  PubMed  Google Scholar 

  172. Bar-Nahum, I. et al. Reduction of nitrous oxide to dinitrogen by a mixed valent tricopper-disulfido cluster. J. Am. Chem. Soc. 131, 2812–2814 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  173. Johnson, B. J., Antholine, W. E., Lindeman, S. V., Graham, M. J. & Mankad, N. P. A one-hole Cu4S cluster with N2O reductase activity: a structural and functional model for CuZ*. J. Am. Chem. Soc. 138, 13107–13110 (2016).

    CAS  Google Scholar 

  174. Esmieu, C. et al. N2O reduction at a dissymmetric {Cu2S}-containing mixed-valent center. Chem. Sci. 5, 4774–4784 (2014).

    CAS  Google Scholar 

Download references

Acknowledgements

Work on P450nor models and related NO complexes by the Lehnert laboratory is supported by a grant from the National Science Foundation (CHE-1464696 to N.L.), which is gratefully acknowledged.

Author information

Authors and Affiliations

  1. Department of Chemistry and Department of Biophysics, University of Michigan, Ann Arbor, MI, USA

    Nicolai Lehnert, Hai T. Dong, Jill B. Harland, Andrew P. Hunt & Corey J. White

Authors
  1. Nicolai Lehnert
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Hai T. Dong
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Jill B. Harland
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Andrew P. Hunt
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Corey J. White
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

All authors contributed equally to the preparation of this manuscript.

Corresponding author

Correspondence to Nicolai Lehnert.

Ethics declarations

Competing interests

The authors declare no competing interests.

Additional information

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Lehnert, N., Dong, H.T., Harland, J.B. et al. Reversing nitrogen fixation. Nat Rev Chem 2, 278–289 (2018). https://doi.org/10.1038/s41570-018-0041-7

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41570-018-0041-7

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing