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Vol. 172, No. 9
JOURNAL OF BACTERIOLOGY, Sept. 1990, p. 4775-4782
0021-9193/90/094775-08$02.00/0
Copyright C) 1990, American Society for Microbiology
Inhibition of Ammonia Monooxygenase in Nitrosomonas europaea
by Carbon Disulfide
MICHAEL R. HYMAN,t CHARLOTTE Y. KIM,T AND DANIEL J. ARPt*
Department of Biochemistry, University of California, Riverside, California 92521
Received 5 March 1990/Accepted 7 June 1990
The resulting hydroxylamine is then oxidized to nitrite, with
the release of four electrons by a complex, multihemecontaining, soluble protein, hydroxylamine oxidoreductase
(11) (HAO):
The major process in microbial nitrification is the oxidation of ammonia to nitrite, followed by the oxidation of
nitrite to nitrate. These reactions are predominantly catalyzed by specialized autotrophic bacteria characterized by
such species as the ammonia-oxidizing bacterium Nitrosomonas europaea and the nitrite-oxidizing bacterium
Nitrobacter winogradskyi, respectively. Although our understanding of the biochemistry and enzymology of individual reactions in the nitrification process is far from complete,
there is a considerable general interest in nitrification because it plays an important role in controlling the balance
between fixed and mobile forms of nitrogen in soil and water
systems. For example, in agriculture, nitrification can lead to
substantial losses of costly ammonia-based fertilizer (and
urea-based fertilizer, which hydrolyzes to ammonia) because
both nitrification products, nitrite and nitrate, are easily
leached from soils by water. In addition, nitrate is the major
substrate for denitrifying bacteria that generate gaseous
products (N20, NO, and N2) which are lost to the atmosphere. These same reactions, while detrimental to agriculture, are regarded as positive benefits in water treatment
regimens, in which the objective is to reduce the nitrogen
content of waste waters (22).
Because it is ammonia-oxidizing bacteria that initiate
nitrification as a whole, there has been considerable research
into the inhibition of this process. Ammonia oxidation by N.
europaea is a two-stage process that is now thought to
involve the initial oxidation of ammonia to hydroxylamine
by a membrane-bound enzyme known as ammonia monooxygenase (15) (AMO):
NH4+
+
XH2
+
02- NH20H
+
H20
+
NH20H + H20-> N02- + 5H+ + 4e(2)
Because hydroxylamine is the only source of reductant
available for AMO activity during steady-state ammonia
oxidation (11, 33), AMO activity is susceptible to inhibition
either through direct effects on the enzyme itself or through
indirect effects on HAO or other components that are
involved in the transfer of electrons to AMO. This unusual
interrelationship is of considerable importance in determining the site of action of ammonia oxidation inhibitors.
One of the oldest known inhibitors of nitrification is
carbon disulfide (CS2), which was first reported by Warrington (31). Even before Warrington's work, CS2 had been
used as a soil fumigant and its effects on nitrification had
been recognized. However, the inhibitory effects of CS2 on
nitrification at concentrations used during soil fumigation
almost certainly arise from its general bacteriocidal qualities.
Powlson and Jenkinson (23) were the first to characterize the
potency of CS2 as a specific nitrification inhibitor and
demonstrated that it effectively inhibits nitrite production
from ammonia at concentrations as low as 0.5 jig/ml. This
finding indicates that ammonia-oxidizing bacteria are the
sensitive agents. Powlson and Jenkinson (23) identified CS2
as the active inhibitor compound released from rubber
stoppers, and they concluded that CS2 was generated as a
breakdown product of sulfur compounds used in the rubbervulcanizing process. Beyond the anecdotal nature of this
discovery, the significance of the potent effects of CS2 on
nitrification is perhaps more widespread than is generally
appreciated, since the action of many other compounds that
inhibit nitrification can also potentially be rationalized in
terms of the mode of action of CS2. These inhibitory
compounds include thiosulfates (18), thiocarbonates (1, 2),
thiocarbamates (12, 18), xanthates (such as potassium ethylxanthate) (18, 29, 30), sulfur-containing amino acids such
H+ + X (1)
* Corresponding author.
t Present address: Laboratory for Nitrogen Fixation Research,
Department of Botany and Plant Pathology, Oregon State University, Corvallis, OR 97331.
t Present address: Medical School, Stanford University, Palo
Alto, CA 94301.
4775
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Carbon disulfide has long been recognized as a potent inhibitor of nitrification, and it is the likely active
component in several nitrification inhibitors suitable for field use. The effects of this compound on Nitrosomonas
europaea have been investigated, and the site of action has been determined. Low concentrations of CS2 (<400
,uM) produced a time-dependent inhibition of ammonia-dependent O2 uptake but did not inhibit hydrazineoxidizing activity. CS2 also produced distinct changes in difference spectra of whole cells. These results suggest
that ammonia monooxygenase (AMO) is the site of action of CS2. Unlike the case for thiourea and acetylene,
saturating concentrations of CS2 did not fully inhibit AMO, and the inhibition resulted in a low but significant
rate of ammonia-dependent 02 uptake. The effects of CS2 were not competitive with respect to ammonia
concentration, and the inhibition by CS2 did not require the turnover of AMO to take effect. The ability of CS2treated cells to incorporate [14C]acetylene into the 28-kilodalton polypeptide of AMO was used to demonstrate
that the effects of CS2 are compatible with a mode of action which involves a reduction of the rate of turnover
of AMO without effects on the catalytic mechanism. It is proposed that CS2 may act on AMO by reversibly
reacting with a suitable nucleophilic amino acid in close proximity to the active site copper.
4776
HYMAN ET AL.
as methionine (20), cystine and cysteine (4, 5, 17), and
several pesticides and fungicides (18, 28), which are all
compounds known to degrade in solution or soils to release
CS2. However, despite widespread interest in inhibitors of
nitrification and the use of CS2 as a specific nitrification
inhibitor in recent field and soil studies (1, 2, 4) neither the
mode nor site of action of CS2 on ammonia-oxidizing bacteria has been established. In this study, we have characterized some of the effects of CS2 on whole cells of N. europaea
and have compared and contrasted these effects with those
produced by acetylene (16) and thiourea (20), two other
well-characterized inhibitors of nitrification.
Calif.).
Growth and harvesting of N. europaea cells. N. europaea
was grown in 1-liter shake flask cultures as described previously (14). Cells were harvested by centrifugation (20,000 x
g for 20 min), and the sedimented cells were suspended in 50
mM sodium phosphate buffer, pH 7.8, containing 2 mM
Mg2+. The cells were then sedimented again, suspended to
approximately 0.2 g (wet weight)/ml in buffer (as described
above), and stored on ice until used (within 3 h of harvest-
ing).
02 uptake measurements. Rates of 02 uptake of cell
suspensions were measured by using a Clark-type oxygen
electrode (Yellow Springs Instrument Co., Yellow Springs,
Ohio) mounted in an all-glass, water-jacketed reaction vessel
(1.6-ml volume). All measurements were made at 30°C; 50
mM sodium phosphate, pH 7.8, containing 2 mM Mg2+ was
used as the buffer in all experiments.
Anaerobic incubation of cells. Incubations of cells under
anaerobic conditions were conducted in an all-glass, micro
reaction vessel. The vessel consisted of a borosilicate glass
lower chamber (1.5 by 0.5 cm; inside diameter, 0.3 cm) with
a female ground-glass neck joint into which a male capillary
section (4 by 0.8 cm; inside diameter, 1 mm) could be
inserted. The volume of the complete vessel was approximately 250 ,ul. Cells were added to fill the lower chamber,
and the capillary section was then inserted. This procedure
forced air bubbles out of the chamber; the displaced cells
were retained in the capillary section and minimized the
diffusion of air into the lower chamber. All additions to the
micro reaction vessel were made from anaerobic stock
solutions, using gas-tight microsyringes.
Spectroscopic studies with whole cells. Difference spectra
obtained with whole cells were recorded with a Beckman
DU-7 spectrophotometer. A whole-cell suspension (1 ml of
0.05 g [wet weight] of cells per ml) was added to a quartz
cuvette (1-cm path length; total volume, 1.2 ml), which was
sealed with a Teflon stopper. A base line was recorded and
memorized. Additions to the cell suspensions were made by
removing the stopper from the cuvette and adding substrates
and inhibitors (10 Pdl) from stock solutions. The cuvette was
then stoppered, inverted twice, and replaced in the spectrophotometer. Spectra were then recorded at the indicated
times.
Fractionation of whole cells and sodium dodecyl sulfatepolyacrylamide gel electrophoresis analysis of membrane proteins. After exposure to mixtures of inhibitors and ["4C]
acetylene, whole cells were sedimented by centrifugation in
a microfuge (12,000 x g for 2 min) and then resuspended to
approximately 0.2 g (wet weight)/ml in 50 mM sodium
phosphate buffer, pH 7.8, containing 2 mM Mg2+. The cells
were then broken by using freezing and thawing cycles and
fractionated into soluble and membrane fractions as described previously (16). After fractionation, 100 p,g of membrane protein was solubilized in microfuge tubes in sample
buffer containing 1% (wt/vol) sodium dodecyl sulfate, 10%
(wt/vol) glycerol, 10% (vol/vol) P-mercaptoethanol, and 62.5
mM Tris hydrochloride, pH 6.8. The samples were solubilized at 60°C for 3 min, and nonsolubilized material was
sedimented by centrifugation. Electrophoresis was conducted at room temperature in 13.5% acrylamide slab gels.
The gels were stained and prepared for fluorography as
described previously (16). Fluorographs were produced by
using Kodak XAR5 X-ray film and a 5-day exposure time.
Protein was determined by using the biuret assay on cells
and membrane fractions that had been solubilized in 3 M
NaOH for 30 min at 65°C. Bovine serum albumin was used
as a standard. The solubility of 02 in air-saturated buffer at
30°C was taken as 230 puM (15), and the solubility of
acetylene in water at 25°C was taken as 42 mM (16).
RESULTS
Since both the oxidation of ammonia to hydroxylamine
(reaction 1) and hydroxylamine oxidation of nitrite (reaction
2) ultimately consume 02, the simplest means by which
these activities can be assayed and the effects of inhibitors
discriminated is by following 02 uptake rates in the presence
of appropriate substrates. In the absence of any inhibitor,
the addition of 10 mM NH4Cl to a whole-cell suspension
resulted in a rapid rate of 02 uptake which, after an initial
short lag phase (<5 s), remained constant until in excess of
95% of the remaining 02 had been consumed (Fig. 1, trace
A). The addition of low concentrations of CS2 (25 to 400 ,uLM)
during the period of steady-state ammonium oxidation resulted in a time-dependent inhibition of oxygen uptake
(Traces B to F). Increases in the CS2 concentration from 25
to 100 p.M increased the rate of inhibition. With higher
concentrations of CS2 (200 and 400 ,uM), the rate of inhibition was the same, which indicated that the system was
saturated with respect to inhibitor concentration. Even with
the highest inhibitor concentration, a residual rate of 02
uptake was observed when the time-dependent effects of
CS2 appeared to have taken full effect. The inhibition of
ammonia-dependent 02 uptake activity by CS2 did not
require the presence of NH4'. The kinetics of the inhibition
caused during preincubation of cells with CS2 were the same
as those observed when CS2 was added during steady-state
rates of ammonia oxidation (data not shown). Although CS2
was added as a solution in DMSO (final concentration in
sample of 0.08 M), DMSO alone did not inhibit either
ammonia or hydrazine oxidation (data not shown).
The site of action of CS2 was investigated in an experiment
in which the effects of acetylene, thiourea, and CS2 on
ammonia and hydrazine oxidation were compared. All three
inhibitors produced time-dependent inhibitions Of 02 uptake
in the presence of 10 mM NH4' (Fig. 2). In all cases, the
addition of 600 p.M hydrazine (an alternative substrate for
HAO) to each incubation 20 min after addition of the
inhibitor led to a stimulation of the rate of 02 uptake. In each
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MATERIALS AND METHODS
Materials. CS2 (spectrophotometric grade, 99+%) was
obtained from Aldrich Chemical Co., Inc. (Milwaukee,
Wis.). Thiourea was obtained from EM Science (Gibbstown,
N.J.). [14C]acetylene was generated from Ba14CO3 and
trapped in dimethyl sulfoxide DMSO, as described by M. R.
Hyman and D. J. Arp (submitted for publication). Ba14CO3
(specific activity, 57.5 mCi/mmol) was obtained from Amersham Corp. (Arlington Heights, Ill.). Electrophoresis materials were supplied by Bio-Rad Laboratories (Richmond,
J. BACTERIOL.
CARBON DISULFIDE INHIBITION
VOL. 172, 1990
case, the rate of 02 uptake in the presence of hydrazine was
not affected by any of the inhibitors tested. In this experiment, both acetylene and thiourea rapidly inactivated ammonia-dependent °2 uptake within 5 min. For both inhibitors, the residual 02 uptake rate was close to zero (Fig. 2,
traces B and C). In contrast, the inhibition caused by CS2
(200 ,uM) took longer to take full effect and resulted in a low
but significant and constant residual rate of 02 uptake (trace
B) that was slightly above 4% of the uninhibited rate
observed with 10 mM NH4+.
1
I
CELLS
The residual activity observed after CS2 treatment was
further investigated by using alternative techniques that did
not rely on 02 uptake measurements. First, the medium
acidification associated with ammonia oxidation (see reactions 1 and 2) was monitored spectrophotometrically, using a
weakly buffered reaction medium containing a pH indicator.
Only a very slight acidification occurred when cells were
incubated in the absence of ammonium (Fig. 3, trace A). In
the presence of 10 mM NH4', a short lag phase was
observed, after which the medium was acidified at a constant
rate until the available 02 in the reaction cuvette was
depleted. Acidification recommenced if the cuvette was
shaken to reintroduce air into the cell suspension (trace B).
Traces C to E show the effects of thiourea, acetylene, and
CS2, respectively, on the time course of medium acidification during steady-state rates of ammonia oxidation. Again,
both acetylene and thiourea fully inhibited ammonia-dependent acidification, whereas CS2-treated cells exhibited significant residual activity after CS2 inhibition had apparently
taken full effect.
The residual level of AMO activity observed after the
addition of CS2 was further confirmed by monitoring the
susceptibility of AMO to inactivation by acetylene after
treatment with either thiourea or CS2. Cells were preincubated for 30 min with either no inhibitor, thiourea, or CS2.
Residual ammonia-oxidizing activity was quantified after 30
and 40 min from initial rates Of 02 uptake in the presence of
10 mM NH4+ to confirm that the effects of each inhibitor had
reached completion during the preincubation period. Equal
concentrations of 14C2H2 (as a solution in DMSO) were then
added to each incubation, and samples of cells were removed after 5, 60, and 180 min. The resulting fluorogram
(Fig. 4) summarizes the effects of thiourea and CS2 on
14C2H2 radiolabel incorporation into the 28-kilodalton (kDa)
membrane-bound polypeptide that has been shown to be the
site of acetylene binding (16). For cells incubated in the
absence of either thiourea or CS2, label incorporation was
nearly complete by the first time point. For cells pretreated
with thiourea, there was no apparent incorporation of label
even after 3 h of exposure. For cells preincubated with CS2,
there was a slow rate of label incorporation over the time
5 min
-
I
"A
N2H4
A
FIG. 2. Effects of acetylene, thiourea, and CS2 on ammonia- and hydrazine-dependent 02-uptake by whole cells of N. europaea. The
experimental conditions were as described for Fig. 1. Once steady-state rates of ammonia-dependent 02 uptake were established, the
reactions were made to 200 ,uM each of (B) CS2, (C) C2H2, and (D) thiourea. Hydrazine was added (to 600 ,uM) to reactions B, C, and D at
the point indicated by the second arrow. (E) Hydrazine (at 600 ,M) replaced NH4Cl at the initiation of the experiment.
Downloaded from http://jb.asm.org/ on November 12, 2017 by guest
FIG. 1. Inhibition by CS2 of ammonia-dependent 02 uptake by
whole cells of N. europaea. 02 uptake was monitored with an
oxygen electrode. In each experiment, 1.6 ml buffer was used and
NH4Cl was added to an initial concentration of 10 mM. The reaction
was initiated by adding whole cells to a final concentration of 0.18
mg/ml. After a steady rate Of 02 uptake was established, CS2 was
added to the electrode chamber at the point indicated by the arrow
to the following concentrations: (A) no addition; (B) 25 ,um; (C) 50
,um; (D) 100 ,uM; (E) 200 ,uM, and (F) 400 ,M. CS2 was added from
a stock solution in DMSO, and additions were equal to or less than
20 ,l.
4777
4778
J. BACTERIOL.
HYMAN ET AL.
Lane Number
l
Io.0
OD
3
2
-
_
-
0
168I
4
B
E
FIG. 3. Effects of acetylene, thiourea, and CS2 on medium
acidification associated with ammonia oxidation. The reaction medium consisted of 5 mM sodium phosphate buffer, pH 7.8, containing 25 mM NaCl and phenol red (5 p,g/ml). For each reaction, 1 ml
of medium was added to a 1.2-ml quartz cuvette, and NH4Cl was
added to a final concentration of 10 mM (traces B to E). The reaction
was initiated by the addition of 20 ,ul of whole-cell suspension of N.
europaea (final protein concentration, 0.22 mg/ml), after which the
cuvette was sealed with a Teflon stopper, inverted twice, and placed
in the spectrophotometer. The traces show the decrease in A,.9 for
cells incubated with (A) no additions, (B) 10 mM NH4Cl alone, (C)
10 mM NH4Cl followed by 100 p.M thiourea, (D) 10 mM NH4Cl
followed by 170 p.M C2H2, and (E) 10 mM NH4Cl followed by 200
p.M CS2. For traces C to E, the inhibitors were added at the break
in the trace. For trace B, the break indicates the point at which the
reaction medium was partially reoxygenated by inverting the stoppered cuvette.
of the experiment which after 3 h was equivalent to
that for cells without prior treatment with any inhibitor.
The experiments described above demonstrate that the
site of action of CS2 is related to the ammonia-oxidizing
enzyme system and that CS2 does not appear to interact with
either HAO or the associated electron transport chain between HAO and the terminal oxidase. However, the results
do not necessarily demonstrate a direct interaction between
CS2 and AMO. Using visible-wavelength difference spectra
of whole cells, Shears and Wood (27) have demonstrated
that a number of compounds which interact with AMO by
different mechanisms all produce absorbance decreases centered at approximately 380 nm in aerobic cell suspensions
incubated in the absence of ammonia. For example, alternative substrates for AMO, such as bromoethane and ethylene,
cause this bleaching effect. We observed a similar effect with
CS2 (Fig. 5). Trace A (Fig. 5) shows the base line recorded
for cells before the addition of any inhibitor. Trace B shows
the effect of 100 pM acetylene after 10 min, and traces C and
D show the time-dependent development of a comparable
spectrum for 100 FM CS2 during a 10-min incubation. The
addition of ammonia to uninhibited cells normally results in
extensive heme reduction (27). However, addition of 10 mM
NH4C1 to the CS2-treated cells did not result in the reduction
of hemes after 1 min (trace E). In contrast, the subsequent
course
7
5
1 80
60
180
(min)
FIG. 4. Time course of the incorporation of [14C]acetylene into
the 28-kDa acetylene-binding polypeptide of AMO for whole cells of
N. europaea preincubated with thiourea and CS2. A 2-ml sample of
a whole-cell suspension (0.1 g [wet weight]/ml) was added to a 9-ml
serum vial that was stoppered with a trifluoroethylene-faced silicone
rubber stopper and aluminum crimp seals. The cells were treated
with either no addition, 200 nmol of thiourea, or 2 ,umol of CS2, and
the vials were placed on an orbital shaker (75 rpm). After 30 min,
cells (20 ,ul) were removed from each vial, and the rate of 02 uptake
in the presence of 10 mM NH4' was determined in an oxygen
electrode. This procedure was repeated 10 min later to ensure
completion of the inhibition by CS2 and thiourea. Then 25 p.l of a
solution of [14C]acetylene in DMSO was added to each vial (1 ,ul =
6.65 x 105 dpm; total [14C]acetylene added, 65 nmol), and NH4Cl
was added to an initial concentration of 2 mM. The vials were then
returned to the shaker. Samples (500 p.l) were then withdrawn from
each vial 5, 60, and 180 min after the addition of the [14C]acetylene.
The samples were mixed with 100 p.J 50 mM sodium phosphate
buffer, pH 7.8, containing 1 mM thiourea. The samples were then
sedimented by centrifugation (12,000 x g for 5 min) and suspended
in thiourea-containing buffer as described above. The cells were
then sedimented again by centrifugation, suspended in thiourea-free
buffer, and frozen in dry ice. The cells were fractionated and
samples were electrophoresed as described in Materials and Methods section. The fluorograph was obtained from sodium dodecyl
sulfate-polyacrylamide gel electrophoresis analysis of membranes
samples from cells incubated with no inhibitor (lanes 1 to 3),
thiourea (lane 4), and CS2 (lanes 5 to 7) and shows the time course
for the incorporation of [14C]acetylene into the 28-kDa polypeptide
of AMO.
addition of 600 ,uM hydrazine resulted in a rapid (within 1
min) and dramatic change that was associated with extensive
heme reduction (trace F). Equivalent results were obtained
in separate experiments using the same sequence of additions to thiourea and acetylene-inhibited cells (data not
shown). These results indicate that CS2, like acetylene and
thiourea, does not inhibit the reduction of hemes by substrates of HAO but does inhibit the reduction of hemes by
ammonia, the substrate of AMO. Control experiments conducted in the absence of inhibitors established that sufficient
oxygen remained after 10 min to support ammonia oxidation
and that the lack of cytochrome reduction after the addition
of NH4Cl to CS2-treated cells (Fig. 5, trace E) was not due to
anaerobic conditions within the assay cuvette.
A potential competitive interaction between ammonia and
CS2 was investigated by conducting a series of 02 uptake
experiments with whole cells in which the ammonium concentration was varied and 02 uptake was inhibited by the
addition of a constant concentration of CS2. Since the
inhibition caused by CS2 is time dependent, the effect of
ammonium concentration was determined by estimating the
time taken for the inhibitor to inhibit the rate of oxygen
uptake by 50%. The results of this experiment and of
comparable experiments with thiourea and acetylene, which
also produced time-dependent inhibitions (Fig. 2), are shown
in Fig. 6. The range of ammonium concentrations used
Downloaded from http://jb.asm.org/ on November 12, 2017 by guest
D
6
__o
Time
C.
5
VOL. 172, 1990
. ~ ~ *_~
CARBON DISULFIDE INHIBITION
A
1.0
,
A 0.8
IF
-I
c
0
cm0.6
a.
4779
0._
0
:5E
0
4
F
0
..O0.2
.-
15
000
--0
Li.
n
10
0
FIG. 5. Effects of acetylene and CS2 on the difference spectra of
whole cells of N. europaea. Difference spectra of whole cells were
obtained as described in Materials and Methods. Shown are the
spectra obtained from whole cells after incubation with (A) no
additions, (B) 200 ,uM C2H2 for 10 min, (C) 200 p.M CS2 for 5 min,
and (D) 200 ,uM CS2 for 10 min. (E) Reaction D, 1 min after addition
of NH4Cl (10 mM); (F) reaction E, 1 min after addition of 600 p.M
hydrazine.
covers concentrations both above and below the apparent
Kinm for NH4 , as determined from initial rates of oxygen
uptake (Fig. 6A). Over the range of ammonium concentrations tested, there' was essentially no change in the t1j2 for
inhibition caused by either thiourea or CS2, implying that
their effects are independent of ammonium (Fig. 6B). In
contrast, the t1l2 for inactivation by acetylene was progressively increased with successive increases in ammonium
concentration.
Beyond competition with respect to ammonia, two additional properties of AMO-specific inhibitors have been used
to distinguish between various modes of action, namely, the
requirement for 02 and the reversibility' of the inhibition.
When whole cells were incubated with CS2 and acetylene
under anaerobic conditions, these compounds caused respective overall losses of 8 and 21% of the ammoniumdependent 02 uptake activity when the cells were assayed
under aerobic conditions after an 80-fold dilution in the
oxygen electrode chamber (Table 1). In contrast, treatment
with thiourea under anaerobic conditions resulted in a loss of
90% of the preinhibited activity. None of the inhibitors
tested inhibited the oxidation of hydrazine. The effects of
CS2 were also found to be partially reversible, and up to 35%
of the uninhibited ammonia-oxidizing activity was recovered
by washing CS2-treated cells in buffer containing NH4'
(Table 2). Lower recoveries of activity were generally ob-
w
I-4
5F
*
* *-
0.25 0.5
1.0
'..
.-
2.5 5.0
10
[NH*]
.
25
50
(mM)
FIG. 6. Effects of ammonium concentration on the half-time of
inhibition of ammonia oxidation by acetylene, thiourea, and CS2. A
series of oxygen electrode incubations was run in which whole cells
(0.18 mg of total protein per nl) were assayed for rates of ammoniadependent 02 uptake in the presence of a range of NH4Cl concentrations between 0.25 and 50 mM. When a steady-state rate Of 02
uptake had been established for each concentration of NH4Cl,
inhibitor (to 200 p.M) was added. The time required for the rate Of 02
uptake to decline to 50% of the preinhibited rate was then determined. (A) Effect of initial NH4Cl concentration on the preinhibited
rate Of 02 uptake; (B) effect of NH4CI concentration on the half-life
of ammonia-dependent 02 uptake activity of cells inhibited with 200
,uM C2H2 (0), 200 ,uM thiourea (0), and 200 ,uM CS2 (A).
served with thiourea-treated cells, and the inclusion of
copper in the washing medium resulted in a slight increase in
the level of recovery compared with other treatments. In
contrast, no recovery of activity was observed with any
treatment for acetylene-treated cells. It was also observed
that ammonia-oxidizing activity increased with prolonged
incubation of CS2-treated cells in the oxygen electrode
chamber. A 6% recovery of residual ammonia-oxidizing
activity was observed 1 min after addition of cells and
NH4Cl to the electrode chamber. However, the activity
increased to 24% at 5 min and 36% at 10 min. The same level
of recovery was observed if CS2-treated cells were added to
the electrode chamber in the absence of ammonium and then
ammonia oxidation was initiated 10 min later by the addition
of 10 mM NH4C1. The levels of recovery reported in Table 2
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20'
I-
4780
HYMAN ET AL.
J. BACTERIOL.
TABLE 1. Effects of incubation of whole cells of N. europaea
with acetylene, thiourea, and CS2 under anaerobic conditionsa
% Inhibition of N2H4-
% Inhibition of ammonia-
Treatment
oxidizing activity
oxidizing activity
0
No addition
3.8
3.3
150 ,uM C2H2
8.1
150 ,uM thiourea
90.3
0
150 P.M CS2
6.7
20.9
a A whole-cell suspension (0.25 ml; 12.5 mg of protein per ml) was added to
the anaerobic incubation vial described in Materials and Methods. NH4Cl was
then added to 1 mM to allow the cells to consume remaining 02 in the vial
represent the activities of cells measured 5 min after addition
to the electrode chamber.
DISCUSSION
In the past, two main experimental approaches have been
used to determine the site of action of nitrification inhibitors.
Since AMO has not been purified beyond a cell-free state,
the combined use of these two approaches provides the most
unambiguous evidence presently available for a direct chemical interaction between an inhibitor and AMO. In 1973,
Hooper and Terry (12) identified several groups of compounds that specifically inhibited ammonia oxidation, by
considering the differential effects of these compounds on
nitrite production from ammonia and hydroxylamine. More
TABLE 2. Effects of washing treatments on the reversibility of
inhibitions caused by acetylene, thiourea, and CS2"
% of activity recovereda
Inhibitor
Buffer
loe
Unwashed
cnrlb
contro
a
b
alone'
a
b
Buffer
plus
100 ~LM
cu2+d
a
b
Buffer
1 mmplus
NH4+d
a
b
No addition
100 100 98 104 98 104 88 104
2 104
Acetylene
2
83
2
88
3
83
96
Thiourea
6 100
16
92 24
84 17
96
22
CS2
113 22
92 29
35 100
A whole-cell suspension of N. europaea (2 ml, 0.1 g [wet weight]/ml) was
a
incubated in 9-ml serum vials sealed with trifluoroethylene-faced silicone
rubber stoppers and aluminum crimp seals. In separate experiments, acetylene (2 ,umol), thiourea (0.1 Lmol), and CS2 (2 I&mol) were added to the vials,
and the reaction mixtures were shaken for 20 min on an orbital shaker (75
rpm). Samples (20 p.l)kwere then removed from each incubation and assayed
for residual ammonia- and hydrazine-oxidizing activities in an oxygen electrode, as described for Table 1. A 1.5-ml sample of the remaining material in
each incubation was then removed, and 0.5 ml was added to each of three
microfuge tubes. The cells were washed by three cycles of centrifugation and
resuspension in various media and finally suspended to a final volume of 0.5
ml in 50 mM sodium phosphate buffer, pH 7.8. Each sample was then assayed
for residual ammonia- and hydrazine-oxidizing activity as described for Table
1. The reported activities were taken 5 min after the addition of cells to the
chamber.
b Column a, ammonia-oxidizing activity; column b, hydrazine-oxidizing
activity.
c Buffer in all cases was 50 mM sodium phosphate, pH 7.8, containing 2 mM
Mg2+.
d Copper was added to buffer as
CuS04; NH4+ was added as NH4Cl.
Downloaded from http://jb.asm.org/ on November 12, 2017 by guest
during a 5-min preincubation. In separate experiments, each inhibitor (to 150
p.M) was then added, and the cells were incubated for a further 15 min. A
sample (20 ,u) of each cell suspension was removed and added to an oxygen
electrode chamber containing 10 mM NH4Cl. The residual rate of ammoniadependent 02 uptake was determined from steady-state rates. Thiourea (100
p.M) was then added to the electrode chamber, and after complete inhibition
of 02 uptake, hydrazine (to 600 FM) was added to the reaction. The residual
hydrazine-oxidizing activity was determined from steady-state rates of 02
uptake.
recently, Shears and Wood (27) have demonstrated that
several classes of AMO-specific inhibitory compounds that
inhibit ammonia oxidation through different mechanisms all
produce spectral changes in whole-cell suspensions that are
compatible with alterations in the oxidation state of the
copper, which is thought to be involved in the catalytic cycle
of AMO. These compounds include (i) alternative substrates
for AMO (e.g., bromoethane and ethylene) (15, 33), (ii) a
metal-binding inhibitor (allylthiourea) (12, 20), and (iii) acetylene, which acts as a suicide substrate for the enzyme (16).
In this study we have presented both comparable kinetic
data, using 02 uptake measurements (Fig. 2) and spectroscopic evidence (Fig. 5), which suggest that CS2 is another
compound which interacts with AMO. What is less clear is
the mode of action of CS2 on this enzyme. Various possible
modes of action are discussed below.
Considerable interest exists in the mammalian toxicology
of CS2 since this compound is used in numerous industrial
processes and represents an industrial hazard because of its
high volatility and toxicity (6, 21). Studies on the effects of
CS2 in mammalian systems have concentrated on cytochrome P-450 monooxygenase systems which, like AMO
from N. europaea, are typically nonspecific enzymes. Large
decreases in the levels of microsomal P-450 are reported to
occur after treatment of test animals with CS2 (9, 10, 13, 21),
and the use of 14CS2 and C35S2 (9, 26) has shown that the
metabolism of CS2 by microsomal fractions involves the
cytochrome P-450-catalyzed oxidation of CS2 to carbonyl
sulfide (COS) and covalent binding of 35S to the enzyme (26).
In the case of AMO, the results obtained in this study
suggest that enzymatic activation of CS2 is not a likely mode
of action for this compound. Despite the apparent requirement for oxygenated conditions, which are required for
AMO activity (Table 1), we did not observe a competitive
interaction between CS2 and ammonia (Fig. 6), as would be
expected for competing oxidizable substrates. In contrast,
both an oxygen requirement (Table 1) and a competitive
interaction with ammonia (Fig. 6) were observed for acetylene, and inactivation by this inhibitor is known to require
the catalytic activity of AMO (16). We also observed that
pretreatment of whole cells with CS2 only delayed and did
not prevent the complete incorporation of [14C]acetylene
into the 28-kD acetylene-binding component of AMO (Fig.
4). If the effects of CS2 involved inactivation of AMO arising
from the products of CS2 oxidation, one expected effect of
pretreatment of cells with CS2 would have been a loWer level
of [14C]acetylene incorporation compared with that observed for cells treated with [14C]acetylene alone. This effect
was not observed (Fig. 4).
An alternative and more likely mode of action for CS2
inhibition of AMO arises from consideration of the chemical
properties of this compound. The chemistry of CS2 is
dominated by its ability to reversibly react with nucleophiles
to produce metal-complexing compounds (24). For example,
CS2 will react with many compounds, including amino acids,
which contain suitable amino, mercapto, and hydroxy
groups to form dithiocarbamates, trithiocarbamates, and
xanthogenates, respectively. These compounds act as chelators of metal ions and have sufficiently high affinities for
both copper and zinc (24) that the reaction of copper salts
with dithiocarbamates generated from CS2 is the basis of
many analytical procedures for CS2 (24). The metal-chelating activity of compounds formed from the reaction of CS2
with amino acids is also argued to be a significant part of the
toxic effect of CS2 on test animals and can account for the
inhibitory effect of CS2 on cytochrome oxidase, monamine
CARBON DISULFIDE INHIBITION
VOL. 172, 1990
B
A
RNH
RN
C=S <-..........--* iSH-
NH2
NH
s
R-NH2 + CS2 = R-NHC
SH
FIG. 7. Differences in the modes of action of thiourea and CS2.
(24, 32). These differences are shown in diagramatic form in
Fig. 7.
On the basis of this interpretation, it would appear that
AMO inhibitors which contain C=S bonds are all metalcomplexing agents that can be further subdivided into compounds which act as true chelating agents (CS2 and compounds that are capable of releasing CS2) and simple metalbinding compounds (thiourea and other alkyl-substituted
thiourea-based compounds). From a structural point of
view, these groups can also be distinguished on the basis of
terminally located C=S groups (CS2, diethyldithiocarbamate,
and potassium ethylxanthate) and internal C=S bonds (thiourea, allylthiourea, thiosemicarbazide, and thioacetamide).
Principal assumptions behind much of the more recent
research into ammonia oxidation has been that AMO is a
copper-containing enzyme (3, 20, 27, 33) and that the mode
of action of compounds such as thiourea involves a copperselective metal-binding reaction (12, 20). These assumptions
are also reflected in this discussion. It is important to note
that one of the main lines of evidence supporting a role for
copper in AMO comes from studies which show metalbinding agents such as thiourea and allythiourea are potent
inhibitors of ammonia oxidation, and it is argued that these
compounds are copper selective (20). It is interesting to note
that outside the field of nitrification, thiourea and alkyl
derivatives are actually regarded as extremely nonspecific
ligands that can form complexes with more than 70 ionic
species (24). More recently, thiourea has been shown to act
as a potent scavenger of both hydroxyl and superoxide
radicals in biological systems (7, 19); monooxygenase-catalyzed reactions may involve these reactive intermediates.
Perhaps the inhibition of AMO by CS2 (and related compounds) provides more convincing evidence for a role for
copper in AMO than is provided by the inhibition by
thiourea and its derivatives. A purified form of AMO and the
establishment of a role for copper in the catalytic cycle of the
enzyme would undoubtedly help in the clarification of these
possibilities.
ACKNOWLEDGMENT
This research was supported by the U.S. Department of Agriculture grant 88-37120-3956.
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through a diversion of the catalytic reaction to a slower, less
efficient pathway (8). In contrast to CS2, thiourea inhibits the
activity of AMO completely. Wood et al. (32) and others
have argued that a likely mode of action for thiourea-based
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reactive thiols. The differences in the modes of action of
thiourea and CS2 may therefore be determined by the ability
of compounds such as dithiocarbamates to act as bidentate
ligands (and thus true chelators), whereas thiourea and
related compounds can serve only as monodentate ligands
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