Department of Microbiology, University of Nijmegen, Toernooidveld 1, 6525 ED Nijmegen, The Netherlands1
Institute for General Botany, Department of Microbiology, University of Hamburg, Ohnhorststraße 18, 22609 Hamburg, Germany2
Author for correspondence: Ingo Schmidt. Tel: +31 24 3652568. Fax: +31 24 3652830. e-mail: i.schmidt{at}sci.kun.nl
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ABSTRACT |
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Keywords: acetylene, nitrogen dioxide, 14C2H2-labelling, NO2, O2-dependent ammonia oxidation
Abbreviations: AMO, ammonia monooxygenase
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INTRODUCTION |
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![]() | (1) |
![]() | (2) |
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
).
![]() | (3) |
The four electrons derived from this reaction enter the AMO reaction (equations 1, 2
), CO2 assimilation and the respiratory chain (Wood, 1986
). The reducing equivalents are transferred to the terminal electron acceptors O2 (oxic conditions) or nitrite (anoxic conditions) (Wood, 1986
; Schmidt & Bock, 1998
). The reduction of nitrite under anoxic conditions leads to the formation of N2, 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 (C2H2) 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 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 14C2H2 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
).
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 NO2 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 N2O4 is the obligatory substrate (oxidant) under both oxic and anoxic conditions.
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METHODS |
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Chemicals.
Unlabelled acetylene gas was obtained from Messer Griesheim (Germany). 14C-labelled acetylene was prepared according to Hyman & Arp (1990) , trapping 14C2H2 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 23 weeks in the dark at 28 °C 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 °C 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 flow-through system, which was constantly flushed with different gas mixtures (100 ml min-1). To study aerobic ammonia oxidation, N2 gas was supplied with 21% O2, 0100 p.p.m. (v/v) NO2 and 010000 p.p.m. (v/v) acetylene. Anaerobic ammonia oxidation was examined in an N2 or He atmosphere supplied with 0100 p.p.m. (v/v) NO2 and 010000 p.p.m. (v/v) acetylene. The N2 gas contained a maximum of 100 p.p.b. oxygen (gas certificate by Messer Griesheim). During the experiments the cell suspension (5 ml, 5x109 cells ml-1) was stirred (800 r.p.m.) to ensure efficient gas transfer with the atmosphere. NO2 consumption and NO production could be calculated on the basis of the different concentrations in the gas inlet and gas outlet. NO and NO2 were detected with a Chemiluminescence Analyser (Eco Physics).
Control experiments were carried out with cell-free medium and heat-sterilized cell suspensions. In further control experiments, chloramphenicol (400 µg ml-1) 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).
Electrophoresis.
SDS-PAGE was carried out with a Mini-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 1·2% 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. 14C2H2-labelled proteins were detected directly after electrophoresis by scintillation autography (fluorography) using X-ray 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 56 d (Bonner & Laskey, 1974 ). The molecular masses of the polypeptides were estimated by comparison with the RF values of molecular mass markers between 14·4 and 97·4 kDa included in each gel.
Analytical procedures.
Measurement of , NH2OH,
, NO and NO2 was carried out as described previously (Schmidt & Bock, 1997
). The protein concentration was determined according to Bradford (1976)
.
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RESULTS |
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Cell samples removed at the indicated times were analysed for labelling with 14C2H2 using SDS-PAGE 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 NO2 did not influence the labelling reaction. The level of label incorporation into the polypeptide increased in a time-dependent 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).
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In further experiments N. eutropha cells were incubated in the presence of 14C2H2, the NO and NO2 concentration was respectively increased up to 1000 p.p.m. and the O2 concentration was adjusted to 100 p.p.m. The labelling kinetics of the 27 kDa polypeptide and inactivation kinetics of the ammonia oxidation were monitored. A lower incorporation activity of 14C2H2 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 NO2 concentration did not affect the labelling and inactivation kinetics. Independent of the NO2 concentration after 15 min, no additional incorporation of 14C2H2 into the polypeptide was detected and aerobic ammonia oxidation was inhibited completely (data not shown).
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DISCUSSION |
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An important observation from the experiments is that anaerobic ammonia oxidation with NO2 (N2O4) as oxidant was not affected by acetylene. N. eutropha cells treated with acetylene oxidized ammonia even under oxic conditions if NO2 was available. The ammonia oxidation activity of acetylene-treated cells in the presence of NO2 was almost the same under both oxic and anoxic conditions. Ammonia oxidation was not detectable in the absence of NO2. Therefore, it is possible to distinguish between NO2-dependent and O2-dependent ammonia oxidation.
One of the most significant observations is that the 27 kDa polypeptide of AMO was not labelled during anoxic NO2-dependent ammonia oxidation. When O2 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 O2.
The results of this study clearly demonstrate that it is necessary to distinguish between NO2-dependent and O2-dependent ammonia oxidation. Further experiments were performed to give evidence of whether the potential oxidizing agents (O2 and N2O4) competed for the same active site. If so, increasing NO2 concentrations should protect the 27 kDa polypeptide against labelling with 14C2H2 and should also protect ammonia oxidation against inhibition. Competition experiments showed that NO2 was able to protect neither the 27 kDa polypeptide against labelling, nor AMO against inactivation. 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 NO2 act at different sites.
The results of this study allowed a modified hypothetical model of ammonia oxidation in N. eutropha. Fig. 4(ac
) shows the model for O2- and NO2-dependent ammonia oxidation. Experiments showed that anaerobic ammonia oxidation is dependent on the presence of the oxidizing agent N2O4 (dimeric form of NO2). NO was produced in stoichiometric amounts and was released into the gas phase. According to these results, Fig. 4(a)
was developed. When NO2 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 NO2 is used as oxidizing agent, NO2 concentrations above 50 p.p.m. are toxic and inhibit ammonia oxidation (Schmidt & Bock, 1997
).
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![]() | (4) |
![]() | (5) |
![]() | (6) |
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, O2) 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. 4ac
) describes both oxic and anoxic ammonia oxidation by N. eutropha. First, NO2 (N2O4) is a suitable oxidizing agent under anoxic conditions, leading to the formation of NO. Second, the addition of NO2 or NO increases ammonia oxidation activity under oxic conditions (Zart & Bock, 1998
). The addition of NOx (NO or NO2) 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 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 O2 and only O2-dependent ammonia oxidation was inhibited. NO2-dependent ammonia oxidation was not affected. If acetylene binds in the presence of O2 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 O2-dependent ammonia oxidation, because the enzyme is now unable to provide the AMO with NO2 (N2O4). The lack of NO oxidizing activity could be compensated by the addition of NO2. Under anoxic conditions NO is an end product, because O2 is not available, but under oxic conditions the acetylene-inhibited enzyme is necessary to produce stoichiometric amounts of NO. Without acetylene inhibition, NO is reoxidized to NO2 and is therefore not detectable in the gas atmosphere of the cell suspensions. Fourth, NO2-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 O2 is necessary for labelling.
Summarizing, the model according to Fig. 4(ac
) (ammonia oxidation with NOx cycle) explains the results of this study. N2O4 is the oxidizing agent for ammonia oxidation. Besides hydroxylamine, NO is produced. Under oxic conditions NO is reoxidized to NO2 (N2O4), again providing the AMO with the oxidizing agent (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 NO2 is added and NO is produced in stoichiometric amounts (ratio of NO2 consumption to NO production about 1:1). The stoichiometry of this reaction is the same that was observed for anaerobic NO2-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, O2 is used to oxidize NO. The product, NO2, is then consumed during ammonia oxidation. The oxygen of hydroxylamine still originates from molecular oxygen, but is incorporated via NO2.
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REFERENCES |
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Received 29 January 2001;
revised 2 April 2001;
accepted 26 April 2001.