Ammonia oxidation by Nitrosomonas eutropha with NO 2 as oxidant is not inhibited by acetylene

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1 Microbiology (2001), 147, Printed in Great Britain Ammonia oxidation by Nitrosomonas eutropha with NO 2 as oxidant is not inhibited by acetylene Ingo Schmidt, 1 Eberhard Bock 2 and Mike S. M. Jetten 1 Author for correspondence: Ingo Schmidt. Tel: Fax: i.schmidtsci.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, Hamburg, Germany The effect of acetylene ( 14 C 2 H 2 ) 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, NO 2 -dependent ammonia oxidation (NO 2 /N 2 O 4 as oxidant) was not affected by acetylene. Further studies gave evidence that the inhibition as well as the labelling reaction were O 2 - dependent. Cells pretreated with acetylene under oxic conditions were unable to oxidize ammonia with O 2 as oxidant. After these cell suspensions were supplemented with gaseous NO 2, ammonia oxidation activity of about 140 µmol NH M 4 (g protein)n1 h N1 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 -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 under anoxic conditions (Rees & Nason, 1966; Schmidt & 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 C, about 70% of NO is in the dimeric form N O. There is evidence that N O instead of NO is used as oxidizing agent (Schmidt & Bock, 1998). NO 2H+2e NH OHH O (1) NN O 2H+2e NH OH2NOH 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 OHH O HNO 4H+4e (3) The four electrons derived from this reaction enter the AMO reaction (equations 1, 2), CO assimilation and the respiratory chain (Wood, 1986). The reducing equivalents 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 SGM 2247

2 I. SCHMIDT, E. BOCK and M. S. M. JETTEN 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). 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 oxic and anoxic conditions. 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 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 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 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, p.p.m. (vv) NO and p.p.m. (vv) acetylene. Anaerobic ammonia oxidation was examined in an N or He atmosphere supplied with p.p.m. (vv) NO and p.p.m. (vv) 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, 510 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). 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). 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 12% 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 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 5 6 d (Bonner & Laskey, 1974). The molecular masses of the polypeptides were estimated by comparison with the R F values of molecular mass markers between 144 and 974 kda included in each gel. Analytical procedures. Measurement of NH+,N 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, were monitored. During the experiment, the ammonia concentration was determined and the labelling of the 27 kda polypeptide (AmoA) with C was analysed. Cell suspensions (510 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 DMSO solution was added to each cell suspension after 8 min (Fig. 1). The ammonia 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). 2248

3 Nitrosomonas eutropha ammonia oxidation 1800 Activity [Ìmol (g protein) 1 h 1 ] Time (min) Fig. 1. Ammonia oxidation activity of N. eutropha cells incubated with 14 C 2 H 2. An aliquot of the 14 C 2 H 2 /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. NO 2 (); oxic atmosphere (21% O 2 ) with 50 p.p.m. NO 2 (); oxic atmosphere (21% O 2 ) without NO 2 (). Fig. 2. Time course of 14 C labelling of polypeptides during incubation of N. eutropha cells with 14 C 2 H 2. 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 NO 2 for 0, 6 and 12 min, respectively (lanes 1 3), in an oxic atmosphere with 50 p.p.m. NO 2 for 0, 6 and 12 min, respectively (lanes 4 6) or in an anoxic atmosphere with 50 p.p.m. NO 2 for 0 and 12 min, respectively (lanes 7 8). 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 of oxygen were added to the system (1% in the 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 (not shown). When the experiment was started in an air 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. There was no effect of acetylene on the hydroxylamine oxidation activity under both oxic and anoxic conditions [about µmol N OH (g protein) h ]. Cell samples removed at the indicated times were analysed for labelling with C 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 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 (510 cells ml ) were initially pretreated in an oxic atmosphere supplied with 500 p.p.m. acetylene (without NO x ) 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 concentrations. The mean values of eight experiments are given 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 experiments, 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, 2249

4 I. SCHMIDT, E. BOCK and M. S. M. JETTEN Table 1. Comparison of the rates of various AMO-dependent activities after treatment of cells with acetylene 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. Gas atmosphere NH+ 4 NO NO 2 O 2 1. Anoxic (He) 0 2. Oxic (air) AnoxicNO 1348 * AnoxicNO OxicNO * OxicNO * About 280 µmol NO (g protein) h was produced. About 15 p.p.m. NO was formed by the gas-phase oxidation of NO by O (2NOO 2NO ; Pires et al., 1994) and was detectable in the gas phase of these experiments. 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. O 2 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 was6%. 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 0005). Without acetylene, the activity remained unchanged between 0 and 250 p.p.m. (error rate 0005). Reaction conditions NO concentration (p.p.m.) Activity in the presence of C Activity without C 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 (15 p.p.m.) were available to the cells (formed by the oxidation of NO by O ). Fig. 3. The effect of NO on 14 C labelling of polypeptides during incubation of ammonia-oxidizing N. eutropha cells with 14 C 2 H 2. The O 2 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 oxic and anoxic conditions (expts 3 and 5), N. eutropha cells oxidized ammonia. The ammonia oxidation activities of these acetylene-pretreated cells of 1348 µmol NH+ (g protein) h (anoxic) and µmol NH+ (g protein) h (oxic) were approximately the same as 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 In further experiments N. eutropha cells were incubated in the presence of C, the NO and NO concentration was respectively increased up to 1000 p.p.m. and the O 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 C 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 additional incorporation of C into the polypeptide was detected and aerobic ammonia oxidation was inhibited completely (data not shown). Influence of O 2 and ammonia concentrations on the inhibitory effect of acetylene O concentrations in the gas atmosphere were varied between 01, 02, 05, 1 and 5%, and ammonia concentrations varied between 02, 1, 5, 10, 50 mm. Acetylene (200 p.p.m.) was added to the gaseous phase above the 2250

5 Nitrosomonas eutropha ammonia oxidation Table 3. Effect of ammonia and O 2 concentrations on ammonia oxidation activity of N. eutropha cells incubated in the presence of acetylene Experiments were performed with cell suspensions of 510 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 8%. According to the Mann Whitney U- Test, the reduced inhibitory effect of acetylene on the ammonia oxidation activity under oxygen-limited conditions is highly significant (error rate of 0005). There is no significant relationship between ammonia concentration and ammonia oxidation activity (error rate of 005). Oxygen concentration (%) Ammonia concentration (mm) 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 oxidant was not affected by acetylene. N. eutropha cells 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 active site. If so, increasing NO concentrations should protect the 27 kda polypeptide against labelling with C H and should also protect ammonia oxidation against inhibition. Competition experiments showed that NO 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 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 oxidation. Experiments showed that anaerobic ammonia oxidation is dependent on the presence of the oxidizing agent N O (dimeric form of NO ). NO was produced in stoichiometric amounts and was released 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 concentrations 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). NN O 2H+2e NH OH2NOH O (4) 2NOO 2NO (N O ) (5) NO 2H+2e NH OHH O (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, 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 ) 2251

6 I. SCHMIDT, E. BOCK and M. S. M. JETTEN (a) (b) (c) Fig. 4. Hypothetical model of ammonia oxidation by N. eutropha (NO x cycle). (a) NO 2 -dependent ammonia oxidation under anoxic conditions, (b) aerobic ammonia oxidation and (c) NO 2 -dependent ammonia oxidation of acetylene-treated cells. Dotted lines: NO or NO 2 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 NO x (NO or NO ) may supply the NO x 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 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 oxidizing activity could be compensated by the addition of NO. Under anoxic conditions NO is an end product, because O 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 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. Summarizing, the model according to Fig. 4(a c) (ammonia oxidation with NO x cycle) explains the results of this study. N O is the oxidizing agent for ammonia oxidation. Besides hydroxylamine, NO is produced. Under oxic conditions NO is reoxidized to NO (N O ), again providing the AMO with the oxidizing agent (NO x cycle). Since detectable NO x concentrations were small, nitrogen oxides seem to cycle in the cell (possibly enzyme-bound) and, therefore, the total amount of NO x 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 is produced in stoichiometric amounts (ratio of NO consumption to NO production about 1:1). The stoichiometry 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. REFERENCES Anderson, K. K. & Hooper, A. B. (1983). O and O are each the source of one O in NO produced from N by Nitrosomonas; N-NMR evidence. FEBS Lett 164,

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