Microbiology (2001), 147, 2247–2253
Printed in Great Britain
Ammonia oxidation by Nitrosomonas eutropha
with NO2 as oxidant is not inhibited by
acetylene
Ingo Schmidt,1 Eberhard Bock2 and Mike S. M. Jetten1
Author for correspondence : Ingo Schmidt. Tel : j31 24 3652568. Fax : j31 24 3652830.
e-mail : i.schmidt!sci.kun.nl
1
Department of
Microbiology, University of
Nijmegen, Toernooidveld
1, 6525 ED Nijmegen,
The Netherlands
2
Institute for General
Botany, Department of
Microbiology, University of
Hamburg, Ohnhorststraße
18, 22609 Hamburg,
Germany
The effect of acetylene (14C2H2) on aerobic and anaerobic ammonia oxidation by
Nitrosomonas eutropha was investigated. Ammonia monooxygenase (AMO)
was inhibited and a 27 kDa polypeptide (AmoA) was labelled during aerobic
ammonia oxidation. In contrast, anaerobic, NO2-dependent ammonia oxidation
(NO2/N2O4 as oxidant) was not affected by acetylene. Further studies gave
evidence that the inhibition as well as the labelling reaction were O2dependent. Cells pretreated with acetylene under oxic conditions were unable
to oxidize ammonia with O2 as oxidant. After these cell suspensions were
supplemented with gaseous NO2, ammonia oxidation activity of about
140 µmol NHM4 (g protein)N1 hN1 was detectable under both oxic and anoxic
conditions. A significantly reduced acetylene inhibition of the ammonia
oxidation activity was observed for cells incubated in the presence of NO. This
suggests that NO and acetylene compete for the same binding site on AMO. On
the basis of these results a new hypothetical model of ammonia oxidation by
N. eutropha was developed.
Keywords : acetylene, nitrogen dioxide, "%C H -labelling, NO - and O -dependent
# #
#
#
ammonia oxidation
INTRODUCTION
Nitrosomonas eutropha is a chemolithotrophic microorganism that obtains its energy for growth from aerobic
or anaerobic ammonia oxidation. The main products
are nitrite under oxic conditions and N , NO and nitrite
# 1966 ; Schmidt
under anoxic conditions (Rees & Nason,
& Bock, 1997). Aerobic (equation 1) and anaerobic
ammonia oxidation (equation 2) are thought to be
initiated by ammonia monooxygenase (AMO) which
oxidizes ammonia to hydroxylamine. O and NO are
assumed to be the oxidizing agents #(Anderson# &
Hooper, 1983 ; Dua et al., 1979 ; Hyman & Wood, 1985 ;
Hyman & Arp, 1993 ; Rees & Nason, 1966 ; Schmidt &
Bock, 1997, 1998). At 25 mC, about 70 % of NO is in the
dimeric form N O . There is evidence that N O# instead
# &
% Bock,
of NO is used# as% oxidizing agent (Schmidt
#
1998).
NH jO j2H+j2e− NH OHjH O
(1)
$
#
#
#
NH jN O j2H+j2e− NH OHj2NOjH O (2)
#
#
$
# %
.................................................................................................................................................
Abbreviation : AMO, ammonia monooxygenase.
The hydroxylamine resulting from ammonia oxidation
is further oxidized to nitrite (equation 3) by hydroxylamine oxidoreductase (HAO) (Arciero & Hooper, 1993 ;
Bergmann et al., 1994 ; Sayavedra-Soto et al., 1994).
NH OHjH O HNO j4H+j4e−
(3)
#
#
#
The four electrons derived from this reaction enter the
AMO reaction (equations 1, 2), CO assimilation and
# reducing equithe respiratory chain (Wood, 1986). The
valents are transferred to the terminal electron acceptors
O (oxic conditions) or nitrite (anoxic conditions)
#
(Wood,
1986 ; Schmidt & Bock, 1998). The reduction of
nitrite under anoxic conditions leads to the formation of
N , resulting in an N-loss of about 45 %.
#
Since AMO has not been characterized beyond cell-free
studies (Schmidt & Bock, 1998 ; Suzuki & Kwok, 1970 ;
Suzuki et al., 1981), much of what is known about the
catalytic enzyme activity has been deduced from inhibitor studies using whole cells. Acetylene (C H ) even
# #
at low concentrations is a specific potent inhibitor
of
ammonia oxidation in Nitrosomonas (Anderson &
Hooper, 1983 ; Dua et al., 1979 ; Hyman & Wood, 1985 ;
Hyman et al., 1994 ; Hynes & Knowles, 1978). Acetylene
0002-4756 # 2001 SGM
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I. S C H M I DT, E. B O C K a n d M. S. M. J E T T EN
has no effect on hydroxylamine oxidation activity at
concentrations sufficient to completely inactivate ammonia oxidation (Hynes & Knowles, 1978). Acetylene is
known to inhibit several metalloenzymes (e.g. nitrogenase, methane monooxygenase). Hyman & Wood
(1985) presented kinetic evidence that acetylene is a
suicidal substrate for AMO. It was proposed that
acetylene inactivates catalytically active AMO as a result
of the attempted oxidation of the triple bond of
acetylene. A reactive intermediate is generated that
covalently binds to the active site of the AMO. The use
of "%C H leads to a covalent modification of a 27 kDa
# #
membrane-bound
polypeptide (Hyman & Arp, 1992).
After inactivation of AMO with acetylene, recovery of
ammonia oxidation activity is dependent on de novo
synthesis of a 27 kDa polypeptide and additional polypeptides which only occurs in the presence of ammonia
(Hyman & Arp, 1995).
(v\v) NO and 0–10 000 p.p.m. (v\v) acetylene. Anaerobic
#
ammonia oxidation
was examined in an N or He atmosphere
#
supplied with 0–100 p.p.m. (v\v) NO and 0–10 000 p.p.m.
#
(v\v) acetylene. The N gas contained a maximum of
#
100 p.p.b. oxygen (gas certificate
by Messer Griesheim).
During the experiments the cell suspension (5 ml, 5i10*
cells ml−") was stirred (800 r.p.m.) to ensure efficient gas
transfer with the atmosphere. NO consumption and NO
#
production could be calculated on the basis of the different
concentrations in the gas inlet and gas outlet. NO and NO
were detected with a Chemiluminescence Analyser (Eco#
Physics).
The main aim of this study was to provide evidence for
the new hypothetical model of aerobic and anaerobic
ammonia oxidation as described in this article. Clearly,
the nitrogen oxides NO and NO are involved in the
metabolism of ammonia oxidizers.#The present study of
the effect of acetylene on aerobic and anaerobic ammonia oxidation by N. eutropha provides data that
N O is the obligatory substrate (oxidant) under both
# %and anoxic conditions.
oxic
Electrophoresis. SDS-PAGE was carried out with a Mini-
METHODS
Organism. Cells of N. eutropha strain N904 (isolated from
cattle manure) were grown and harvested as described
previously (Schmidt & Bock, 1997). The strain is kept in the
culture collection of the Institut fu$ r Allgemeine Botanik
(University of Hamburg, Germany).
Chemicals. Unlabelled acetylene gas was obtained from
Messer Griesheim (Germany). "%C-labelled acetylene was
prepared according to Hyman & Arp (1990), trapping "%C H
# #
with DMSO. An aliquot of this solution was added to the
cell suspensions. Control experiments were performed using
acetylene-free DMSO.
Medium and growth conditions. Precultures were grown
aerobically in 1 l Erlenmeyer flasks containing 600 ml mineral
medium (Schmidt & Bock, 1997). The cultures were grown for
2–3 weeks in the dark at 28 mC without stirring or shaking.
The purity of the cultures was checked by inoculation of
organic liquid and agar-based media, containing (per litre
distilled water) : 1 g yeast extract, 1 g peptone, 2 g Casamino
acids and 2 g beef extract. Furthermore, the cultures were
examined by phase-contrast microscopy. With the absence of
cell growth after 3 weeks incubation at 28 mC under oxic and
anoxic conditions and uniform cell composition the cultures
were judged to be pure.
Experimental design. All experiments were carried out as
described previously using Clark-type Oxygen Electrode Units
(DW1 ; Bachofer) as reaction vessels (vol. 10 ml) (Schmidt &
Bock, 1997). The electrode unit was equipped with a gas flowthrough system, which was constantly flushed with different
gas mixtures (100 ml min−"). To study aerobic ammonia
oxidation, N gas was supplied with 21 % O , 0–100 p.p.m.
#
#
Control experiments were carried out with cell-free medium
and heat-sterilized cell suspensions. In further control experiments, chloramphenicol (400 µg ml−") was added to prevent
de novo synthesis of proteins. Hydroxylamine oxidation
activities of N. eutropha cells were examined under the
conditions described above, but ammonia was replaced by
hydroxylamine as substrate (2 mM).
Protean II Cell vertical gel electrophoresis chamber (Bio-Rad).
Prior to loading, the samples (200 µg protein) were mixed with
an equal volume of a mercaptoethanol solution (1 %) with 2 %
SDS, 20 % sucrose and 1n2 % Tris at room temperature. The
SDS-PAGE gel consisted of a 12 % acrylamide resolving gel
and a 4 % stacking gel (every lane was loaded with 100 µg
protein). The gels were stained with Coomassie brilliant blue.
"%C H -labelled proteins were detected directly after electro# #
phoresis by scintillation autography (fluorography) using Xray films. The gels were dehydrated in DMSO, drenched in a
solution of 2,5-diphenyloxazole (PPO) in DMSO, dried and
exposed to X-ray film for 5–6 d (Bonner & Laskey, 1974). The
molecular masses of the polypeptides were estimated by
comparison with the RF values of molecular mass markers
between 14n4 and 97n4 kDa included in each gel.
Analytical procedures. Measurement of NH+, NH OH, NO−,
%
#
#
NO and NO was carried out as described previously (Schmidt
#
& Bock, 1997). The protein concentration was determined
according to Bradford (1976).
RESULTS
Effect of acetylene on aerobic and anaerobic
ammonia oxidation by N. eutropha
The initial experiments aimed at determining the inhibitory effect of acetylene on both aerobic and anaerobic ammonia oxidation by N. eutropha, i.e. the
time-dependent change of ammonia oxidation activity
in the presence of "%C H , were monitored. During the
# #concentration was determined
experiment, the ammonia
and the labelling of the 27 kDa polypeptide (AmoA)
with "%C H was analysed. Cell suspensions (5i10*
cells ml−"#) #were incubated in gas-tight incubation
chambers in three different atmospheres. First, an oxic
atmosphere (air) ; second, an oxic atmosphere (air)
supplied with 50 p.p.m. NO ; and third, an anoxic
#
atmosphere (He) supplied with
50 p.p.m. NO . An
aliquot of "%C H \DMSO solution was added to# each
# #after 8 min (Fig. 1). The ammonia
cell suspension
oxidation activities without added acetylene (0–8 min)
were in the same order of magnitude as reported
previously (Schmidt & Bock, 1997 ; Zart et al., 2000).
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Nitrosomonas eutropha ammonia oxidation
Activity [Ìmol (g protein)–1 h–1]
1800
1500
1200
900
600
300
5
10
15
20
Time (min)
25
30
.................................................................................................................................................
Fig. 1. Ammonia oxidation activity of N. eutropha cells
incubated with 14C2H2. An aliquot of the 14C2H2/DMSO
solution was added to each cell suspension after 8 min. The cell
suspensions were incubated under different atmospheres :
anoxic atmosphere with 50 p.p.m. NO2 (
) ; oxic atmosphere
(21 % O2) with 50 p.p.m. NO2 (5) ; oxic atmosphere (21 % O2)
without NO2 (#).
Under oxic conditions, activity was about 1425 µmol
NH+ (g protein)−" h−", under oxic conditions with
%
50 p.p.m.
NO activity was 1655 µmol NH+ (g
protein)−" h−" #and under anoxic conditions %with
50 p.p.m. NO it was 145 µmol NH+ (g protein)−" h−".
#
%
After 8 min acetylene was added. The ammonia oxidation activity of cell suspensions incubated under
anoxic conditions in an He atmosphere with 50 p.p.m.
NO was not influenced by acetylene (Fig. 1). The cells
were# not affected by the inhibitory effect of acetylene
and their activity remained unchanged [145 µmol NH+
(g protein)−" h−"]. When instead of NO small amounts%
# (1 % in the
of oxygen were added to the system
atmosphere), cells became completely inactive within
20 min. Under oxic conditions (air) without NO ,
#
ammonia oxidation activity disappeared completely
within 12 min in a time-dependent process. After adding
50 p.p.m. NO , ammonia oxidation started again with
an activity of# about 127 µmol NH+ (g protein)−" h−"
% started in an air
(not shown). When the experiment was
atmosphere supplied with 50 p.p.m. NO , the addition
of acetylene led to a significant decrease# in ammonia
oxidation activity from 1655 to about 130 µmol NH+ (g
%
protein)−" h−" (Fig. 1). The remaining activity was nearly
the same as under anoxic conditions [130 to 145 µmol
NH+ (g protein)−" h−"]. Obviously, acetylene did
not %affect ammonia oxidation with NO as oxidant.
# hydroxylThere was no effect of acetylene on the
amine oxidation activity under both oxic and
anoxic conditions [about 1970p50 µmol NH OH
#
(g protein)−" h−"].
Cell samples removed at the indicated times were
analysed for labelling with "%C H using SDS-PAGE
# #
.................................................................................................................................................
Fig. 2. Time course of 14C labelling of polypeptides during
incubation of N. eutropha cells with 14C2H2. The position of the
30 kDa molecular mass marker is shown on the left side of the
fluorogram. Cells were incubated in an oxic atmosphere
without NO2 for 0, 6 and 12 min, respectively (lanes 1–3), in an
oxic atmosphere with 50 p.p.m. NO2 for 0, 6 and 12 min,
respectively (lanes 4–6) or in an anoxic atmosphere with
50 p.p.m. NO2 for 0 and 12 min, respectively (lanes 7–8).
and fluorography. The fluorogram (Fig. 2) shows that a
polypeptide with a molecular mass of about 27 kDa was
labelled under oxic conditions. The presence of NO did
#
not influence the labelling reaction. The level of label
incorporation into the polypeptide increased in a timedependent process and corresponded to the decreasing
ammonia oxidation activity. When cells were incubated
under anoxic conditions, the 27 kDa polypeptide was
not labelled. After the addition of 1 % oxygen to the
atmosphere, labelling started immediately (data not
shown).
Influence of nitrogen oxides on ammonia oxidation
of acetylene-treated N. eutropha cells
Previous work showed that the supplementation of both
NO and NO led to increasing nitrification activities of
N. eutropha #under oxic conditions (Zart et al., 2000).
Since ammonia oxidation with NO as oxidizing agent
was not influenced by acetylene, the#influence of NO on
acetylene-treated cells was investigated. The cells
(5i10* cells ml−") were initially pretreated in an oxic
atmosphere supplied with 500 p.p.m. acetylene (without
NOx) to inhibit aerobic O -dependent ammonia oxidation completely (15 min).# The ammonia oxidation
activity of these cells was determined under different
conditions with regard to O , NO and NO concen# experiments#are given
trations. The mean values of eight
in Table 1.
The results obtained with acetylene-treated cells show a
strong correlation between ammonia oxidation and the
presence of NO . As expected from previous experi#
ments, N. eutropha
cells were not able to oxidize
ammonia under anoxic conditions (expt 1) in the
absence of the oxidizing agent NO . Experiment 2, with
#
cells incubated under oxic conditions
without NO ,
#
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I. S C H M I DT, E. B O C K a n d M. S. M. J E T T EN
Table 1. Comparison of the rates of various
AMO-dependent activities after treatment of cells
with acetylene
Table 2. Effect of NO on the inhibition of ammonia
oxidation activity of N. eutropha by acetylene incubated
in the presence of 100 p.p.m. O2
.................................................................................................................................................
.................................................................................................................................................
The steady-state rates of ammonia, NO, NO and O
#
#
consumption are expressed in µmol (g protein)−" h−". Nitrogen
oxides were added at a concentration of 50 p.p.m. Cells were
pretreated with acetylene in an oxic atmosphere for 15 min.
–, Substrate was not added to the gas atmosphere.
Activity was determined after 15 min of acetylene exposure
time. As a control, cells were incubated without acetylene.
Activity is expressed as µmol NH+ (g protein)−" h−". The
%
standard deviation for 12 replicated experiments wasp6 %.
According to the Mann–Whitney U-Test, the increasing
ammonia oxidation activity with increasing NO concentrations
up to 250 p.p.m. is highly significant (error rate of 0n005).
Without acetylene, the activity remained unchanged between
0 and 250 p.p.m. (error rate 0n005).
Gas atmosphere
1.
2.
3.
4.
5.
6.
Anoxic (He)
Oxic (air)
AnoxicjNO
#
AnoxicjNO
OxicjNO
#
OxicjNO
NH+4
NO
NO2
O2
0
0
134p8
0
139p11
5p2
–
–
*
0
*
8p4
–
–
277p 18
–
275p19
†
–
0
–
–
0
6p2
* About 280 µmol NO (g protein)−" h−" was produced.
† About 1n5 p.p.m. NO was formed by the gas-phase oxidation
#
of NO by O (2NOjO
2NO ; Pires et al., 1994) and was
#
#
#
detectable in the gas phase of these experiments.
.................................................................................................................................................
Fig. 3. The effect of NO on 14C labelling of polypeptides
during incubation of ammonia-oxidizing N. eutropha cells with
14
C2H2. The O2 concentration was adjusted to 100 p.p.m. The
position of the 30 kDa molecular mass marker is shown on the
left side of the fluorogram. Cells were incubated without NO
for 0, 7 and 15 min, respectively (lanes 1–3) or in an atmosphere
with 1000 p.p.m. NO for 0, 7 and 15 min, respectively (lanes
4–6).
shows that the pretreatment with acetylene was successful and O -dependent ammonia oxidation was
#
inactivated completely.
If NO was added under both
# 3 and 5), N. eutropha
oxic and anoxic conditions (expts
cells oxidized ammonia. The ammonia oxidation activities of these acetylene-pretreated cells of 134p8 µmol
NH+ (g protein)−" h−" (anoxic) and 139p11 µmol NH+
%
%
−" h−" (oxic) were approximately the same as
(g protein)
in non-treated cells [anoxic : 145 µmol NH+ (g
%
protein)−" h−"]. The ability of the cells to oxidize
ammonia with NO as oxidant was not affected by
acetylene treatment #under both oxic and anoxic conditions. During NO -dependent ammonia oxidation,
#
NO was consumed. Ammonia
and NO were consumed
#
in a ratio of approximately 1 : 2. The #same ratio was
Reaction conditions
NO concentration (p.p.m.)
0
Activity in the presence of
CH
# #
Activity without C H
# #
50 100 250 400 700 1000
0 12 27 49 21
0
0
103 107 104 108 43
0
0
detectable between ammonia consumption and NO
production. Although the cells did not oxidize ammonia
under anoxic conditions with NO (expt 4), demonstrating that NO is not a suitable oxidant for ammonia
oxidation, a low ammonia oxidation activity was
detectable when NO was added under oxic conditions
(expt 6). This result may reflect the fact that small
amounts of NO (1n5 p.p.m.) were available to the cells
#
(formed by the oxidation
of NO by O ).
#
In further experiments N. eutropha cells were incubated
in the presence of "%C H , the NO and NO con# #increased up to 1000 #p.p.m.
centration was respectively
and the O concentration was adjusted to 100 p.p.m.
# kinetics of the 27 kDa polypeptide and
The labelling
inactivation kinetics of the ammonia oxidation were
monitored. A lower incorporation activity of "%C H in
# #
the 27 kDa polypeptide (Fig. 3) and a significantly
reduced inhibition of ammonia oxidation activity (Table
2) was observed for cells which were incubated in a gas
atmosphere with NO concentrations up to 250 p.p.m.
When the atmosphere was supplied with NO concentrations between 300 and 1000 p.p.m., NO had an
inhibitory effect on ammonia oxidation (Table 2, control
without acetylene). The NO concentration did not
#
affect the labelling and inactivation
kinetics. Independent of the NO concentration after 15 min, no ad#
ditional incorporation
of "%C H into the polypeptide
# #
was detected and aerobic ammonia
oxidation was
inhibited completely (data not shown).
Influence of O2 and ammonia concentrations on the
inhibitory effect of acetylene
O concentrations in the gas atmosphere were varied
#
between
0n1, 0n2, 0n5, 1 and 5 %, and ammonia concentrations varied between 0n2, 1, 5, 10, 50 mM. Acetylene
(200 p.p.m.) was added to the gaseous phase above the
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Nitrosomonas eutropha ammonia oxidation
Table 3. Effect of ammonia and O2 concentrations on
ammonia oxidation activity of N. eutropha cells
incubated in the presence of acetylene
.................................................................................................................................................
Experiments were performed with cell suspensions of 5i10*
cells ml−". Activity is expressed as µmol NH+ (g protein)−" h−".
%
At 1 and 5 % O no activity was detected at any ammonia
#
concentration. The standard deviation for four replicated
experiments was p8 %. According to the Mann–Whitney UTest, the reduced inhibitory effect of acetylene on the ammonia
oxidation activity under oxygen-limited conditions is highly
significant (error rate of 0n005). There is no significant
relationship between ammonia concentration and ammonia
oxidation activity (error rate of 0n05).
Oxygen concentration ( %)
0n1
0n2
0n5
1n0
5n0
Ammonia concentration (mM)
0n2
1
5
10
50
927
543
17
0
0
923
550
18
0
0
931
552
15
0
0
937
551
21
0
0
934
563
24
0
0
cell suspension. The ammonia oxidation activity was
measured after 10 min incubation. Obviously, the rate
of inhibition fell as the O concentration decreased
#
(Table 3). In contrast to earlier
reports (Hyman &
Wood, 1985), statistical analysis did not show a
significant protective effect of high ammonia concentrations against acetylene inhibition.
DISCUSSION
On the basis of the results presented above, a new
hypothetical model of ammonia oxidation by N.
eutropha was developed. A new complex role of
nitrogen oxides on the metabolism of these organisms is
proposed. The major aim of this study was to investigate
the effects of acetylene on aerobic and anaerobic
ammonia oxidation.
An important observation from the experiments is that
anaerobic ammonia oxidation with NO (N O ) as
#
# %cells
oxidant was not affected by acetylene. N. eutropha
treated with acetylene oxidized ammonia even under
oxic conditions if NO was available. The ammonia
oxidation activity of #acetylene-treated cells in the
presence of NO was almost the same under both oxic
#
and anoxic conditions.
Ammonia oxidation was not
detectable in the absence of NO . Therefore, it is
possible to distinguish between NO #-dependent and O #
#
dependent ammonia oxidation.
One of the most significant observations is that the
27 kDa polypeptide of AMO was not labelled during
anoxic NO -dependent ammonia oxidation. When O
was added # the labelling of this polypeptide started#
immediately. An influence of the ammonia concentration on the labelling reaction was not observed. It is
interesting to note that not only is an active AMO
(Hyman & Wood, 1985) necessary for labelling the
27 kDa polypeptide, but also the presence of O .
#
The results of this study clearly demonstrate that it is
necessary to distinguish between NO -dependent and
#
O -dependent ammonia oxidation. Further
experiments
#
were performed to give evidence of whether the potential
oxidizing agents (O and N O ) competed for the same
#
#NO
% concentrations should
active site. If so, increasing
protect the 27 kDa polypeptide# against labelling with
"%C H and should also protect ammonia oxidation
# # inhibition. Competition experiments showed
against
that NO was able to protect neither the 27 kDa
# against labelling, nor AMO against inpolypeptide
activation. It is surprising that NO protected aerobic
ammonia oxidation against the inhibitory effect of
acetylene. The most plausible interpretation of these
observations is that NO and acetylene compete for the
same active site, while NO and NO act at different sites.
#
The results of this study allowed a modified hypothetical
model of ammonia oxidation in N. eutropha. Fig. 4(a–c)
shows the model for O - and NO -dependent ammonia
# showed# that anaerobic amoxidation. Experiments
monia oxidation is dependent on the presence of the
oxidizing agent N O (dimeric form of NO ). NO was
# %
# released
produced in stoichiometric
amounts and was
into the gas phase. According to these results, Fig. 4(a)
was developed. When NO was added under anoxic
#
conditions, ammonia was oxidized
and hydroxylamine
occurred as an intermediate, while NO was formed as
an end product (see equation 4 below). Hydroxylamine
is further oxidized to nitrite (Schmidt & Bock, 1998).
Although NO is used as oxidizing agent, NO concen#
trations above# 50 p.p.m. are toxic and inhibit ammonia
oxidation (Schmidt & Bock, 1997).
The situation is more complex under oxic conditions. In
the presence of O , the NO produced can be oxidized to
NO . Therefore,# only small amounts of NO were
#
detectable
in the gas phase of N. eutropha cell
suspensions. According to the model (Fig. 4b, equation
4), N O is the oxidizing agent under oxic conditions.
# %
Hydroxylamine
and NO are produced as intermediates.
While hydroxylamine is further oxidized to nitrite, NO
is (re)oxidized to NO (N O ) (equation 5).
# # %
NH jN O j2H+j2e− NH OHj2NOjH O (4)
$
# %
#
#
2NOjO
2NO (N O )
(5)
#
# # %
(6)
NH jO j2H+j2e− NH OHjH O
#
#
$
#
The sum of equations 4 and 5, given in equation 6, has
been described before as the reaction of aerobic ammonia oxidation (Anderson & Hooper, 1983 ; Dua et al.,
1979 ; Hyman & Wood, 1985 ; Hyman & Arp, 1993 ;
Rees & Nason, 1966), but in agreement with the new
hypothetical model, though the total consumption rates
(ammonia, O ) and production rates (hydroxylamine as
intermediate)#remain unchanged, the mechanism of the
reaction is different.
Several lines of evidence suggest that this hypothetical
model (Fig. 4a–c) describes both oxic and anoxic
ammonia oxidation by N. eutropha. First, NO (N O )
# # %
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I. S C H M I DT, E. B O C K a n d M. S. M. J E T T EN
labelling. This result indicates that NO and acetylene
may compete for the same binding site at the 27 kDa
polypeptide. Furthermore, the 27 kDa polypeptide was
only labelled in the presence of O and only O dependent ammonia oxidation was #inhibited. NO#dependent ammonia oxidation was not affected. #If
acetylene binds in the presence of O at the NO-binding
#
site, it may inactivate the NO oxidizing
function of the
AMO (Fig. 4c) as a result of the attempted oxidation of
the acetylene triple bond. The mechanism as such was
proposed earlier, but the ammonia-binding site was
suspected to be the binding site for acetylene (Hyman &
Arp, 1992). Inactivating the NO oxidizing function of
the AMO leads to inhibition of aerobic O -dependent
ammonia oxidation, because the enzyme is #now unable
to provide the AMO with NO (N O ). The lack of NO
# # % by the addition
oxidizing activity could be compensated
of NO . Under anoxic conditions NO is an end product,
# O is not available, but under oxic conditions
because
#
the acetylene-inhibited
enzyme is necessary to produce
stoichiometric amounts of NO. Without acetylene
inhibition, NO is reoxidized to NO and is therefore not
detectable in the gas atmosphere of#the cell suspensions.
Fourth, NO -dependent ammonia oxidation under
#
anoxic conditions
is not affected by acetylene. While
AMO is active under these conditions, the 27 kDa
polypeptide is not labelled. Obviously, not only an
active AMO, but also the presence of O is necessary for
#
labelling.
(a)
(b)
(c)
.................................................................................................................................................
Fig. 4. Hypothetical model of ammonia oxidation by N.
eutropha (NOx cycle). (a) NO2-dependent ammonia oxidation
under anoxic conditions, (b) aerobic ammonia oxidation and (c)
NO2-dependent ammonia oxidation of acetylene-treated cells.
Dotted lines : NO or NO2 added to the gas atmosphere to act as
substrates.
is a suitable oxidizing agent under anoxic conditions,
leading to the formation of NO. Second, the addition of
NO or NO increases ammonia oxidation activity under
oxic# conditions (Zart & Bock, 1998). The addition of
NOx (NO or NO ) may supply the NOx cycle with
additional substrate# (Fig. 4b), increasing the amount of
oxidant available to the cells and finally increasing
ammonia oxidation activity. Third, increasing NO
concentrations protected the 27 kDa polypeptide from
Summarizing, the model according to Fig. 4(a–c) (ammonia oxidation with NOx cycle) explains the results of
this study. N O is the oxidizing agent for ammonia
# % hydroxylamine, NO is produced.
oxidation. Besides
Under oxic conditions NO is reoxidized to NO (N O ),
# agent
# %
again providing the AMO with the oxidizing
(NOx cycle). Since detectable NOx concentrations were
small, nitrogen oxides seem to cycle in the cell (possibly
enzyme-bound) and, therefore, the total amount of NOx
per cell is expected to be low. The addition of acetylene
leads to an inhibition of ammonia oxidation. The cells
restart ammonia oxidation when NO is added and NO
# (ratio of NO
is produced in stoichiometric amounts
consumption to NO production about 1 : 1). The stoi-#
chiometry of this reaction is the same that was observed
for anaerobic NO -dependent ammonia oxidation.
#
This hypothetical model is in good agreement with the
described mechanisms of aerobic ammonia oxidation
(Anderson & Hooper, 1983 ; Dua et al., 1979 ; Hyman &
Wood, 1985 ; Hyman & Arp, 1993 ; Rees & Nason,
1966). According to the new model, O is used to oxidize
#
NO. The product, NO , is then consumed
during
#
ammonia oxidation. The oxygen of hydroxylamine still
originates from molecular oxygen, but is incorporated
via NO .
#
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.................................................................................................................................................
Received 29 January 2001 ; revised 2 April 2001 ; accepted 26 April 2001.
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