J. Biol. Chem., Vol. 255, Issue 11, 5027-5030, Jun, 1980
13N,15N isotope and kinetic evidence against hyponitrite as an intermediate in dentrification
TC Hollocher, E Garber, AJ Cooper and RE Reiman
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.
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