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 Downloaded from http://jb.asm.org/ on November 12, 2017 by guest 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 Downloaded from http://jb.asm.org/ on November 12, 2017 by guest 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 Downloaded from http://jb.asm.org/ on November 12, 2017 by guest 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. LITERATURE CITED 1. Ashworth, J., G. G. Briggs, A. A. Evans, and J. Matula. 1977. Inhibition of nitrification by Nitrapyrin, carbon disulphide, and trithiocarbonate. J. Sci. Food Agric. 28:673-683. 2. Ashworth, J., A. Penny, F. V. Widdowson, and G. G. Briggs. 1980. The effects of injecting nitrapyrin ('N-serve'), carbon disulfide or trithiocarbonates with aqueous ammonia, on yield and %N of grass. J. Sci. Food Agric. 31:229-237. 3. Bedard, C., and R. Knowles. 1989. Physiology, biochemistry, and specific inhibitors of CH4, NH4', and CO oxidation by methanotrophs and nitrifiers. Microbiol. Rev. 53:68-84. 4. Bremner, J. M., and L. G. Bundy. 1974. Inhibition of nitrification in soils by volatile sulfur compounds. Soil Biol. Biochem. 6:161-165. 5. Bremner, J. M., and C. G. Steele. 1978. Role of microrganisms Downloaded from http://jb.asm.org/ on November 12, 2017 by guest oxidase, and tyrosine oxidase (Cu containing) and on lactate dehydrogenase, carbonic anhydrase, and alcohol dehydrogenase (Zn requiring) (6). In the case of AMO, the formation of chelating species arising from the reversible reaction of CS2 with a suitably positioned amino acid close to an active-site copper would account not only for the time dependency and specificity of the reaction but also for the spectroscopic effects, the lack of competition between ammonia and CS2, and the apparent lack of a requirement for the turnover of AMO during the inhibition process. Compounds that contain C=S bonds represent an important class of potent inhibitors of ammonia oxidation by N. europaea. These compounds include CS2, thiourea, allylthiourea, thiosemicarbazide, thioacetamide, potassium ethylxanthate, and diethyldithiocarbamate. It has been argued that the inhibitor mechanism is largely dictated by functional groups (3, 25); therefore, the presence of a C=S bond in all of the compounds listed above tnight be taken to indicate that a proposed mechanism for one compound is generally applicable to all compounds of this group. In fact, the similarity between the inhibitory effects of these compounds on ammonia oxidation and purified copper-containing enzymes represents a significant proportion of the available evidence for a role for copper in ammonia oxidation. However, it is clear from our results that the effects of CS2 are significantly different than those that we observed in parallel experiments with thiourea. This finding suggests that the mechanisms of these two compounds are different and that the relationship between functional group and inhibitor mechanism is more complex than it first appeared. The significance of the differences between these two inhibitors are considered more thoroughly below. The effects of CS2 are superficially distinguished from those of thiourea in that CS2 produces a relatively slow, time-dependent inhibition which results in a residual activity greater than the endogenous rate of AMO-independent respiration. In the case of partial inhibitions, the analysis of 02 uptake rates cannot clearly distinguish the contributions made by either ammonia oxidation or hydroxylamine oxidation. The more significant difference between the effects of these inhibitors is therefore the residual activity of AMO in CS2-treated cells. This activity is most clearly demonstrated by the ability of CS2-treated cells to incorporate [14C] acetylene to the same level as do non-CS2-treated cells (Fig. 4), albeit at a slower rate. This result indicates two points. First, prolonged incubation with CS2 beyond that characterized in our short-term 02 uptake experiments (Fig. 1 and 2) does not lead to a complete inhibition of AMO. Second, since a complete inhibition does not occur, the effects of CS2 appear to be equally applied to AMO, and the incomplete inactivation by CS2 does not arise from mixed populations of inhibited and uninhibited enzyme. In contrast to the fully inhibitory effects of thiourea, the mode of action of CS2 probably therefore involves a process in which the turnover of AMO is dramatically slowed but not halted. Potentially, incomplete inhibition can arise from allosteric inhibition or 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 compounds involves their ability to tautomerize and form reactive thiols. 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The effects of carbon disulphide on rate liver microsomal mixed-functional oxidases in vivo and in vitro. Biochem. J. 188:107-112. 22. Painter, H. A. 1986. Nitrification in the treatment of sewage and waste-waters, p. 185-211. In J. I. Prosser (ed.), Nitrification. IRL Press, Oxford. 23. Powlson, D. S., and D. S. Jenkinson. 1971. Inhibition of nitrification by carbon disulphide from rubber bungs. Soil Biol. Biochem. 3:267-269. 24. Reid, E. E. 1963. Organic chemistry of bivalent sulfur, vol. 4 and 5. Chemical Publishing Co., Inc., New York. 25. Sahrawat, K. L., and D. R. Keeney. 1985. Perspectives for research on development on nitrification inhibitors. Commun. Soil Sci. Plant Anal. 16:517-524. 26. Savolainen, H., J. Jarvisalo, and H. Vainiio. 1977. Specific binding of CS2 metabolites to microsomal proteins in rat liver. Acta Pharmacol. Toxicol. 41:94-96. 27. Shears, J. H., and P. M. Wood. 1985. Spectroscopic evidence for a photosensitive oxygenated state of ammonia mono-oxygenase. 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