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CLAUDE FROMAGEOT AND JACQUES C. SENEZ
possible that the intermediary compound corresponding to the degree
of oxidation (NOH) is not formed in the free state but only as an enzyme-substrate complex or in bound, organic form.
Few studies have been made and very little is known about the
enzymes which catalyze the oxidation of ammonia to nitrite. Hofman
and Lees (76) showed that the well-known inhibition of Nitrosomonas
by allylthiourea takes place specifically in the first metabolic stage, i.e.,
during oxidation of ammonium ion to hydroxylamine, and does not
affect the oxidation of NH 2 OH to nitrite. They further reported that the
enzyme system which catalyzes the first reaction requires the presence
of a metal of unidentified nature. Recently, Imshenetskii et al. (78) prepared extracts of cells of Nitrosomonas europea which oxidize ammonia
and hydroxylamine to nitrite in vitro. The enzyme is relatively heatstable, retaining its activity after brief exposure to 100° but not after
maintenance of this temperature for 30 minutes.
Specific "reductases" have been extracted from various heterotrophic
bacteria, molds, and higher plants; these enzymes catalyze reactions
which are the inverse of that carried out by Nitrosomonas, i.e., the reduction of nitrite and hydroxylamine to ammonia. Spencer et al. (79)
and Zucker and Nason (80) demonstrated that these enzymes are
metalloflavoproteins coupled functionally with pyridine-nucleotide-dependent dehydrogenases. They are related to nitrate reductase; the
similarity is particularly striking with the nitrite reductase of Neurospora crassa (79), in which the metal has been shown to be molybdenum, as in nitratase. It may be thought a priori that the enzymes which
enable Nitrosomonas to oxidize ammonia are reversible reductases or
closely related enzymes. In support of this hypothesis, Klausmeier and
Bard (81) made cell-free extracts of Bacillus subtilis containing an alcohol dehydrogenase which catalyzed the reduction of NH 2 OH to ammonia in the presence of ethanol, glucose-6-phosphate, or reduced coenzyme I (DPNH). They reported the reduction by these preparations
of oxidized diphosphopyridine nucleotide (DPN) in the presence of
ammonia and considered the reversibility of the reaction as proven.
However Roussos et al. (82) could not confirm this latter point and
showed that the observed reduction of DPN by ammonia was in reality
a nonenzymatic process without physiological significance.
The equilibrium constants of the reactions catalyzed by nitrite and
hydroxylamine reductases (79, 80) favor the formation of ammonia and
the reoxidation of pyridine nucleotides so strongly that their physiological function in the inverse direction, toward nitrification, is most unlikely. This is equally true of nitrate reductase; it is, therefore, reason-
CLAUDE FROMAGEOT AND JACQUES C. SENEZ
possible that the intermediary compound corresponding to the degree
of oxidation (NOH) is not formed in the free state but only as an enzyme-substrate complex or in bound, organic form.
Few studies have been made and very little is known about the
enzymes which catalyze the oxidation of ammonia to nitrite. Hofman
and Lees (76) showed that the well-known inhibition of Nitrosomonas
by allylthiourea takes place specifically in the first metabolic stage, i.e.,
during oxidation of ammonium ion to hydroxylamine, and does not
affect the oxidation of NH 2 OH to nitrite. They further reported that the
enzyme system which catalyzes the first reaction requires the presence
of a metal of unidentified nature. Recently, Imshenetskii et al. (78) prepared extracts of cells of Nitrosomonas europea which oxidize ammonia
and hydroxylamine to nitrite in vitro. The enzyme is relatively heatstable, retaining its activity after brief exposure to 100° but not after
maintenance of this temperature for 30 minutes.
Specific "reductases" have been extracted from various heterotrophic
bacteria, molds, and higher plants; these enzymes catalyze reactions
which are the inverse of that carried out by Nitrosomonas, i.e., the reduction of nitrite and hydroxylamine to ammonia. Spencer et al. (79)
and Zucker and Nason (80) demonstrated that these enzymes are
metalloflavoproteins coupled functionally with pyridine-nucleotide-dependent dehydrogenases. They are related to nitrate reductase; the
similarity is particularly striking with the nitrite reductase of Neurospora crassa (79), in which the metal has been shown to be molybdenum, as in nitratase. It may be thought a priori that the enzymes which
enable Nitrosomonas to oxidize ammonia are reversible reductases or
closely related enzymes. In support of this hypothesis, Klausmeier and
Bard (81) made cell-free extracts of Bacillus subtilis containing an alcohol dehydrogenase which catalyzed the reduction of NH 2 OH to ammonia in the presence of ethanol, glucose-6-phosphate, or reduced coenzyme I (DPNH). They reported the reduction by these preparations
of oxidized diphosphopyridine nucleotide (DPN) in the presence of
ammonia and considered the reversibility of the reaction as proven.
However Roussos et al. (82) could not confirm this latter point and
showed that the observed reduction of DPN by ammonia was in reality
a nonenzymatic process without physiological significance.
The equilibrium constants of the reactions catalyzed by nitrite and
hydroxylamine reductases (79, 80) favor the formation of ammonia and
the reoxidation of pyridine nucleotides so strongly that their physiological function in the inverse direction, toward nitrification, is most unlikely. This is equally true of nitrate reductase; it is, therefore, reason-