Free Standard AU & NZ Shipping For All Book Orders Over $80!
Register      Login
Functional Plant Biology Functional Plant Biology Society
Plant function and evolutionary biology
Shopping Cart: ( empty )
You are here: Home > Journals > FP > FP05284
RESEARCH ARTICLE
Contents Vol 33(6)

Isotopic fractionation by plant nitrate reductase, twenty years later

Guillaume Tcherkez A B C and Graham D. Farquhar B
+ Author Affiliations
- Author Affiliations

A Laboratoire d’Ecophysiologie Végétale, Bâtiment 362, Université Paris XI, 91405 Orsay, France.

B Environmental Biology Group, Research School of Biological Sciences, Australian National University, GPO Box 475, Canberra, ACT 2601, Australia.

C Corresponding author. Email: guillaume.tcherkez@ese.u-psud.fr

Functional Plant Biology 33(6) 531-537 https://doi.org/10.1071/FP05284
Submitted: 24 November 2005  Accepted: 10 March 2006   Published: 1 June 2006

Abstract

Plant nitrate reductase, the enzyme that reduces nitrate (NO3–) to nitrite (NO2–), is known to fractionate N isotopes, depleting nitrite in 15N compared with substrate nitrate. Nearly 20 years ago, the nitrogen isotope effect associated with this reaction was found to be around 1.015. However, the relationships between the isotope effect and the mechanism of the reaction have not yet been examined in the light of recent advances regarding the catalytic cycle and enzyme structure. We thus give here the mathematical bases of the 14N / 15N and also the 16O / 18O isotope effects as a function of reaction rates. Enzymatic nitrate reduction involves steps other than NO3– reduction itself, in which the oxidation number of N changes from +V (nitrate) to +III (nitrite). Using some approximations, we give numerical estimates of the intrinsic N and O isotope effects and this leads us to challenge the assumptions of nitrate reduction itself as being a rate-limiting step within the nitrate reductase reaction, and of the formation of a bridging oxygen as a reaction intermediate.


Acknowledgments

GT thanks the Australian Department of Education, Science and Training for its financial support through an Endeavour Post-doctoral Fellowship of the Australian Education International Department. GF acknowledges the support of the Australian Research Council.


References


Amberger A, Schmidt HL (1987) Natürliche Isotopengehalte von Nitrat als Indikatoren für dessen Herkunft. Geochimica et Cosmochimica Acta 51, 2699–2705.
| Crossref | GoogleScholarGoogle Scholar | open url image1

Barber MJ, Notton BA (1990) Spinach nitrate reductase. Plant Physiology 93, 537–540.
| PubMed |
open url image1

Berti PJ (1999) Determining transition states from kinetic isotope effects. Methods in Enzymology 138, 355–397. open url image1

Bigeleisen J (1949) The relative reaction velocities of isotopic molecules. Journal of Chemical Physics 17, 665. open url image1

Bigeleisen J, Wolfsberg M (1958) Theoretical and experimental aspects of isotope effects in chemical kinetics. Advances in Chemical Physics I, 15–76. open url image1

Brown LL, Drury JS (1967) Nitrogen isotope effects in the reduction of nitrate, nitrite and hydroxylamine to ammonia. I. In sodium hydroxide solution with Fe (II). Journal of Chemical Physics 46, 2833–2837.
| Crossref | GoogleScholarGoogle Scholar | open url image1

Campbell WH (1999) Nitrate reductase structure, function and regulation: bridging the gap between biochemistry and physiology. Annual Review of Plant Physiology and Plant Molecular Biology 50, 277–303.
| Crossref | GoogleScholarGoogle Scholar | PubMed | open url image1

Cleland WW (2005) The use of isotope effects to determine enzyme mechanisms. Archives of Biochemistry and Biophysics 433, 2–12.
| Crossref | GoogleScholarGoogle Scholar | PubMed | open url image1

Craig JA, Holm RH (1989) Reduction of nitrate to nitrite by molybdenum-mediated atom transfer: a nitrate reductase analogue reaction system. Journal of the American Chemical Society 111, 2111–2115.
| Crossref | GoogleScholarGoogle Scholar | open url image1

Farquhar GD , Barbour MM , Henry BK (1998) Interpretation of oxygen isotope composition of leaf material. In ‘Stable isotopes, integration of biological, ecological and geochemical processes’. (Ed. H Griffiths) pp. 27–62. (Bios Scientific Publishers: Oxford)

Fischer K, Barbier GG, Hecht HJ, Mendel RR, Campbell WH, Schwarz G (2005) Structural basis of eukaryotic nitrate reduction: crystal structure of the nitrate reductase active site. The Plant Cell 17, 1167–1179.
| Crossref | GoogleScholarGoogle Scholar | PubMed | open url image1

George GN, Mertens JA, Campbell WH (1999) Structural changes induced by catalytic turn-over at the molybdenum site of Arabidopsis nitrate reductase. Journal of the American Chemical Society 121, 9730–9731.
| Crossref | GoogleScholarGoogle Scholar | open url image1

Hille R, Rétey J, Bartlewski-Hof U, Reichenbecher W, Shink B (1999) Mechanistic aspects of molybdenum-containing enzymes. FEMS Microbiology Reviews 22, 489–501. open url image1

Kohl DH, Shearer G (1980) Isotopic fractionation associated with symbiotic N2 fixation and uptake of NO3– by plants. Plant Physiology 66, 51–56.
| PubMed |
open url image1

Ledgard SF, Woo KC, Bergersen FJ (1985) Isotopic fractionation during reduction of nitrate and nitrite by extracts of spinach leaves. Australian Journal of Plant Physiology 12, 631–640. open url image1

Lillo C, Kazazaic S, Ruoff P, Meyer C (1997) Characterization of nitrate reductase from light and dark-exposed leaves. Plant Physiology 114, 1377–1383.
| PubMed |
open url image1

Lin H, Zhao Y, Ellingson BA, Pu J, Truhlar DG (2005) Temperature dependence of carbon-13 kinetic isotope effects of importance to climate change. Journal of the American Chemical Society 127, 2830–2831.
| Crossref | GoogleScholarGoogle Scholar | PubMed | open url image1

Melander L (1960) ‘Isotope effects on reaction rates.’ (Ronald Press Company: New York)

Meyer MP, DelMonte AJ, Singleton DA (1999) Reinvestigation of the isotope effects for the Claisen and aromatic Claisen rearrangements: the nature of the Claisen transition states. Journal of the American Chemical Society 121, 10865–10874.
| Crossref | GoogleScholarGoogle Scholar | open url image1

Needoba JA, Sigman DM, Harrison PJ (2004) The mechanism of isotope fractionation during algal nitrate assimilation as illuminated by the 15N / 14N of intracellular nitrate. Journal of Phycology 40, 517–522. open url image1

National Institute of Standards and Technology (2005) NIST Chemistry Webbook. http://webbook.nist.gov/chemistry [verified 23 March 2006]

O’Leary MH (1980) Determination of heavy atom isotope effects on enzyme-catalyzed reactions. Methods in Enzymology 64, 83–104.
| PubMed |
open url image1

Olleros-Izard T (1983) Kinetische Isotopeneffekte der Arginase und Nitratreduktase Reaktion: ein Betrag zur Aufklärung der entsprechenden Reaktionmechanismen. PhD thesis, Technischen Universität München, Freising-Weihenstephan.

Paneth P (1995) Theoretical calculations of heavy-atom isotope effects. Computers & Chemistry 19, 231–240.
| Crossref | GoogleScholarGoogle Scholar | PubMed | open url image1

Robinson R (2001) δ15N as an integrator of the nitrogen cycle. Trends in Ecology & Evolution 16, 153–162.
| Crossref | GoogleScholarGoogle Scholar | open url image1

Saunders L (1986) Kinetic isotope effects. In ‘Investigation of rates and mechanisms of reactions, Part I, C’. (Ed. F Bernosconi) pp. 211–255. (Wiley and Sons: New York)

Schmidt HL, Medina R (1991) Possibilities and scope of the double isotope effect method in the elucidation of mechanisms of enzyme catalyzed reactions. Isotopenpraxis 27, 1–4. open url image1

Schmidt S, Dennison WC, Moss GJ, Stewart GR (2004) Nitrogen ecophysiology of Heron Island, a sub-tropical coral cay of the Great Barrier Reef, Australia. Functional Plant Biology 31, 517–528.
| Crossref | GoogleScholarGoogle Scholar | open url image1

Schrader N, Fischer K, Theis K, Mendel RR, Schwarz G, Kisker C (2003) The crystal structure of plant sulfite oxidase provides insights into sulfite oxidation in plants and animals. Structure 11, 1251–1263.
| Crossref | PubMed |
open url image1

Schultz BE, Gheller SF, Muetterties MC, Scott MJ, Holm RH (1993) Molybdenum-mediated oxygen atom transfer: an improved analog reaction system of the molybdenum oxotransferases. Journal of the American Chemical Society 115, 2714–2722.
| Crossref | GoogleScholarGoogle Scholar | open url image1

Shearer G, Kohl DH (1986) N2 fixation in field settings: estimations based on natural 15N abundance. Australian Journal of Plant Physiology 13, 699–756. open url image1

Sims LB , Lewis DE (1984) Bond order methods for calculating isotope effects in organic reactions. In ‘Isotope effects, recent development in theory and experiment’. (Eds E Buncel, CC Lee) pp. 161–259. (Elsevier: New York)

Skipper L, Campbell WH, Mertens JH, Lowes DJ (2001) Pre-steady-state kinetic analysis of recombinant Arabidopsis NADH-nitrate reductase. Journal of Biological Chemistry 276, 26995–27002.
| Crossref | GoogleScholarGoogle Scholar | PubMed | open url image1

Solomonson LP, Barber MJ (1990) Assimilatory nitrate reductase: functional properties and regulation. Annual Review of Plant Physiology and Plant Molecular Biology 41, 225–253.
| Crossref | GoogleScholarGoogle Scholar | open url image1

Taylor RD, Spence JT (1975) Reduction of nitrate by diammonium oxopentachloromolybdate (V) in dimethylformamide. Inorganic Chemistry 14, 2815–2818.
| Crossref | GoogleScholarGoogle Scholar | open url image1

Taylor RD, Todd PG, Chasteen ND, Spence JT (1979) Reduction of nitrate by monomeric molybdenum (V) complexes in dimethylformamide. Inorganic Chemistry 18, 44–48.
| Crossref | GoogleScholarGoogle Scholar | open url image1

Tcherkez G, Farquhar GD (2005) Carbon isotope effect predictions for enzymes involved in the primary carbon metabolism of plant leaves. Functional Plant Biology 32, 277–291.
| Crossref | GoogleScholarGoogle Scholar | open url image1

Urey HC (1947) The thermodynamic properties of isotopic substances. Journal of the Chemical Society 1, 562–581. open url image1

Van Hook A (1971) Kinetic isotope effects: introduction and discussion of the theory. In ‘Isotope effects in chemical reactions’. (Eds CJ Collins, NS Bowman) pp. 20–35. (Van Nostrand Reinhold Co.: New York)

Weast R (1971) ‘Handbook of chemistry and physics.’ (The Chemical Rubber Co.: Cleveland)

Weighardt K, Woeste M, Roy PS, Chaudhuri P (1985) Kinetics and mechanism of nitrate reduction by dimeric aquamolybdenum(III) complex in aqueous solution: direct evidence for an oxo-group transfer. Journal of the American Chemical Society 107, 8276–8277.
| Crossref | GoogleScholarGoogle Scholar | open url image1

Werner RA, Schmidt HL (2002) The in vivo nitrogen isotope discrimination among organic plant compounds. Phytochemistry 61, 465–484.
| Crossref | GoogleScholarGoogle Scholar | PubMed | open url image1

Yakir D (2002) Global enzymes: sphere of influence. Nature 416, 795.
| Crossref | GoogleScholarGoogle Scholar | PubMed | open url image1

Yoneyama T, Ito O, Engelaar WMHG (2003) Uptake, metabolism and distribution of nitrogen in crop plants traced by enriched and natural 15N: progress over the last 30 years. Phytochemistry Reviews 2, 121–132.
| Crossref | GoogleScholarGoogle Scholar | open url image1