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13N,15N isotope and kinetic evidence against hyponitrite as an intermediate in dentrification.

Publication ,  Journal Article
Hollocher, TC; Garber, E; Cooper, AJ; Reiman, RE
Published in: J Biol Chem
June 10, 1980

13N- and 15N-labeling experiments were carried out with Paracoccus denitrificans, grown anaerobically on nitrate, to determine whether hyponitrite might be an obligatory intermediate in denitrification and a precursor of nitrous oxide. From experiments designed to trap [13N]- or [15N,15N]hyponitrite by dilution into authentic hyponitrite it was calculated that the intracellular concentration of a presumptive hyponitrite pool must be less than 0.4 mM. In order for a pool of this size to turn over rapidly enough to handle the flux of nitrogen during dentrifucation, the spontaneous rate of hyponitrite dehydration must be enhanced by a factor of several thousand through enzyme catalysis. Cell extracts failed to catalyze this reaction under a variety of conditions. It is concluded that hyponitrite cannot be an intermediate in dentrification. In addition, the assimilation of inorganic nitrogen was studied in P. denitrificans using 13N as tracer. At low concentrations (less than 10(-8) M) of labeled nitrate and nitrite 5 to 10% of the label was assimilated into non-volatile metabolites and 90 to 95% was reduced to N2. Similarly, with 15 mM [13N]nitrate, 5% of the label went into metabolites and 95% to N2. High pressure liquid chromatography analysis of the labeled metabolites indicated that the major pathway for assimilation of inorganic nitrogen in P. denitrificans under these conditions is through ammonia incorporation via the aspartase reaction.

Duke Scholars

Published In

J Biol Chem

ISSN

0021-9258

Publication Date

June 10, 1980

Volume

255

Issue

11

Start / End Page

5027 / 5030

Location

United States

Related Subject Headings

  • Paracoccus denitrificans
  • Nitrous Oxide
  • Nitrogen Radioisotopes
  • Nitrogen Isotopes
  • Nitrites
  • Nitrates
  • Kinetics
  • Biochemistry & Molecular Biology
  • Amino Acids
  • 11 Medical and Health Sciences
 

Citation

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Hollocher, T. C., Garber, E., Cooper, A. J., & Reiman, R. E. (1980). 13N,15N isotope and kinetic evidence against hyponitrite as an intermediate in dentrification. J Biol Chem, 255(11), 5027–5030.
Hollocher, T. C., E. Garber, A. J. Cooper, and R. E. Reiman. “13N,15N isotope and kinetic evidence against hyponitrite as an intermediate in dentrification.J Biol Chem 255, no. 11 (June 10, 1980): 5027–30.
Hollocher TC, Garber E, Cooper AJ, Reiman RE. 13N,15N isotope and kinetic evidence against hyponitrite as an intermediate in dentrification. J Biol Chem. 1980 Jun 10;255(11):5027–30.
Hollocher, T. C., et al. “13N,15N isotope and kinetic evidence against hyponitrite as an intermediate in dentrification.J Biol Chem, vol. 255, no. 11, June 1980, pp. 5027–30.
Hollocher TC, Garber E, Cooper AJ, Reiman RE. 13N,15N isotope and kinetic evidence against hyponitrite as an intermediate in dentrification. J Biol Chem. 1980 Jun 10;255(11):5027–5030.

Published In

J Biol Chem

ISSN

0021-9258

Publication Date

June 10, 1980

Volume

255

Issue

11

Start / End Page

5027 / 5030

Location

United States

Related Subject Headings

  • Paracoccus denitrificans
  • Nitrous Oxide
  • Nitrogen Radioisotopes
  • Nitrogen Isotopes
  • Nitrites
  • Nitrates
  • Kinetics
  • Biochemistry & Molecular Biology
  • Amino Acids
  • 11 Medical and Health Sciences