Proc. Natl. Acad. Sci. USA Vol. 79, pp. 5871-5875, October 1982 Biochemistry

180 isotope shift in 15N NMR analysis of biological N-oxidations: H20-N02 exchange in the ammonia-oxidizing bacterium Nitrosomonas (hydroxylamine/nitrite/oxygen exchange) KRiSTOFFER K. ANDERSSON*, STEPHEN B. PHILSONt, AND ALAN B. HOOPER*t *Department of Genetics and Cell Biology, University of Minnesota, St. Paul, Minnesota 55108; and tDepartment of Chemistry, University of Minnesota, Minneapolis, Minnesota 55455 Communicated by W. D. McElroy, July 12, 1982

ABSTRACT The 180/160 shifts in '5N NMR were deter- hydrazine (an inhibitor of hydroxylamine oxidation) has recent- mined for nitrite (0.13 ppm or 4.2 Hz at 7.05 T) and nitrate (0.056 ly been demonstrated by Dua et aL (4) and Hollocher et aL (6) ppm or 1.7 Hz at 7.05 T) at neutral pH. The technique, which utilizing mass spectrometric analysis. allows clear differentiation between 160 and 180 derivatives of 15N NMR is an ideal technique for identifying the origin of '5N, was used to assess the source of oxygens in nitrite produced the two oxygens ofnitrite. The great potential of 15N NMR spec- by oxidation ofammonia in Nitrosomonas. The two oxygens of ni- troscopy-until recently obscured by the low abundance of15N trite produced by cell-catalyzed oxidation of ammonia or hydrox- (0.365%) and low sensitivity ("5N has a nuclear moment only ylamine had the 160/180 isotope composition of water. Nitroso- 1/10 that of the proton)-has become realizable with the in- monas is shown to catalyze the rapid exchange ofoxygen between Fourier nitrite and water. The exchange reaction required the concomi- troduction of higher fields and transform techniques tant oxidation ofammonia. The amount ofnitrite exchanged could (for reviews, see refs. 7 and 8). Because '5N has spin 1/2, the exceed the amount of ammonia oxidized by a factor of3. This ex- spectra normally have narrow lines, while the chemical shift change explains previous difficulties in the determination of the range is =800 ppm, giving potentially very great resolution. source ofnitrite oxygen in ammoniaoxidation. When cells oxidized In the present work we observe the incorporation of 180 ['5N]ammonia in the presence of a great excess of exogenous into N02 using the secondary isotope effect-a shift in the 15N [14Njnitrite, 20% of one oxygen in the resulting ['5N]nitrite was resonance of N02- when 180 is substituted for 160 The 180 derived from dioxygen. Dioxygen is apparently the source of at isotope-induced shift in 15N NMR has recently been measured least one oxygen in nitrite produced by Nitrosomonas. by Van Etten and Risley (9). We now report the utilization of 15N(180) NMR in biology. The experimental protocol itself is Nitrosomonas species catalyze the oxidation of ammonia to ni- not substantially different from earlier ones which utilized mass trite as the sole source of energy for growth. Oxidation of am- spectroscopy (5). However, the NMR method has the advantage monia does not occur under anaerobic conditions (for review, that it does not require the synthesis ofvolatile derivatives (con- see ref. 1). At low oxygen concentrations significant amounts version of N02- to CO2)'for gas analysis; after incubation, bac- of other oxides of nitrogen (N20 and NO) are produced in ad- teria are merely spun down and the supernatant ofthe reaction dition to nitrite (2). mixture analyzed directly in a NMR tube. Oxidation ofammonia is believed to involve hydroxylamine, Our initial efforts to demonstrate the biological incorporation NH20H, as intermediate; Nitrosomonas can oxidize hydrox- of 1802 into N02 failed. Nitrite invariably assumed the isoto- ylamine to nitrite, and hydroxylamine is detected during am- pic composition ofthe H20. We found acell-catalyzed exchange monia oxidation in the presence of hydrazine (3, 4). The overall reaction of N02- oxygen atoms with those ofH20. The reaction reaction can be described as follows: required the concomitant oxidation of ammonia or hydroxyl- amine to nitrite. The rapid biological exchange reaction explains '/202 '/202 the difficulty in assessing the source of the second oxygen in HN02 + 2H nitrite (5). By adding an excess of N02 before the oxidation NH3NHA.NH2OH-"NH20H of [F5N]ammonia it was possible to obtain incorporation of ox- jr HN02 + 4HW. ygen from 02, providing the best evidence to date that dioxygen H20 is the source of at least one of the oxygen atoms in ammonia- Whether oxygen atoms added in the conversion ofammonia derived nitrite in cells ofNitrosomonas. to nitrite come from molecular oxygen or from water has not been clearly shown. Knowing the source ofthe nitrite oxygens METHODS is central to an understanding of the in vivo system and of the Growth of Cells. Nitrosomonas europaea cells (Schmidt enzyme hydroxylamine oxidoreductase (EC 1.7.3.4), which cat- strain) were grown and harvested as described in 90-liter batch alyzes oxidation of hydroxylamine to nitrite (1). Previous anal- cultures (10). The cells were collected in the logarithmic phase ysis involving mass spectrometry achieved an incorporation of of growth when the nitrite concentration of the media had 7% of one 1 O-atom into nitrite when 1802 was provided (5), reached 20-35 mM. Cells were washed two times with 50 mM indicating that at least one oxygen of nitrite probably comes phosphate buffer at pH 7.5 and resuspended in 0.5 M phos- from molecular oxygen. Incorporation ofmolecular oxygen into phate/10 mM carbonate buffer, pH 8.0, to a concentration of hydroxylamine during ammonia oxidation in the presence of 800 mg ofwet weight per ml. Cells were used the same day or the day after harvest; they remained active at least 1 wk after The publication costs ofthis article were defrayed in part by page charge harvesting. payment. This article must therefore be hereby marked "advertise- ment" in accordance with 18 U. S. C. §1734 solely to indicate this fact. t To whom reprint requests should be addressed. 5871 Downloaded by guest on September 30, 2021 5872 Biochemistry: Andersson et al. Proc. Nad Acad. Sci. USA 79 (1982) Isotope Experiments. A'15-ml centrifuge tube was filled with 18 02or 602 at atmospheric pressure and sealed with a serum stopper. A volume of.4 or 0.6 ml of 1602-saturated or Ar-satu- rated 0.25 M phosphate/5 mM carbonate buffer, pH 8.0 or 7.5 (for ammonia oxidation or for hydroxylamine oxidation, respec- tively), was transferred into the sealed tube with a gastight Hamilton syringe. For ammonia oxidation (which was linear as afunction oftime), ammonium sulfate and sometimes potassium or sodium nitrite were present in the degassed buffer. For hy- droxylamine oxidation, hydroxylamine hydrochloride in de- gassed 0.5 M phosphate buffer at pH 6.5 was added by a gas- tight Hamilton syringe into the sealed tube every 10 min. Each addition increased the concentration of hydroxylamine by 6 mM. To start an experiment, the bacterial suspension was added V v I. by Hamilton syringe to afinal cell concentration of40 mg ofwet VWI .,I I-. weight per ml. Tubes were then placed on a rotating wheel at 233.4 233.2 233.0 232.8 232.6 0.2 0.0 -0.2 -0.4 room temperature. Samples (2-10 skd) were withdrawn by a gas- Chemical shift, ppm tight Hamilton syringe for measurement. ofnitrite production. The reaction was stopped by placing the centrifuge tube in an FIG. 1 180/160 derivatives of nitriteandnitrate resolvedby high- resolution 15N NMR. (A) Solution of 100 mM [1PN]nitrite in 0.25 M ice bath for 3 min; cells were then sedimented at 10,000 x g phosphate/0.5 mM carbonate/6% H2180, pH 7.5; 180 scans,. 12-mm for 5 min. The supernatant was analyzed by NMR the same day tube (4 ml). (B) Solution of [15N]nitrite in 0.25 M phosphate/5 mM or was frozen in liquid nitrogen and subsequently thawed prior carbonate/10% H2180, pH 7.0; 724 scans, 12-mm tube. to NMR analysis. The pH value at the end of the experiment was never lower than 6.7. For overnight NMR collections, the changing 15N02- in acidic solution with H2180 (90% 180) and pH was increased by NaOH to 9 to prevent the chemical ex- then adding a comparable amount of '5N602- (the leftmost change reaction (9). peak). The peaks are well resolved, with a separation of 0. 13 Nitrite Analysis. Nitrite was estimated by diazotization (11). ppm (4.2 Hz at 7.05 T). The spectrum required 180 accumu- Synthesis of ['5N'8O]Nitrite and Nitrate. Synthesis of lations and was obtained in less than 1/2 hr. 15N"02- was by the method ofAnbar et aL (12); Na15N02 was Fig. 1B shows the isotope effect seen in 15NO3 resonance dissolved in H2180, acidified with HC1, and reacted for 3 days synthesized in an analogous way. All four oxygen combinations in a sealed tube. The reaction was stopped by the addition of are well resolved and each peak is separated by 0.056 ppm (1.7 a 0.25 M phosphate solution at pH 8. Synthesis of'5N803- was Hz at 7.05 T). The 180 isotopic shift in [15N]nitrate is seen to by the method of Jordan and Bonner (13). A solution of be only 43% as large as the effect in nitrite, reflecting the sub- Na'5NO2 and K'5NO3 in 40% H2180 was acidified with HC1. stantially different electronic structures of the two ions. After reaction for 8 days in a sealed tube, the solution was neu- Nitrite Produced by Oxidation of Ammonia or Hydroxyl- tralized with 40% H2180/0.25 M phosphate solution. amine by Nitrosomonas Does Not Contain Oxygen from Diox- NMR. NMR spectra were made with a Nicolet NT-300 spec- ygen. Incubation of cells with 100 mM 15NH4 for 2 hr in the trometer at 30.42 MHz (7.05'T). To maximize resolution some presence of 100% 1802 (gas), as sole source of 1802, resulted in measurements of the isotope effect were made in 5-mm tubes the formation of40 mM nitrite. The NMR spectrum of the re- (0.5 ml solution), whereas the entire 15N spectrum was normally sulting nitrite (the solid line in Fig. 2A) contained a single peak. obtained with 12-mm tubes (=4-ml sample) for maximum sen- This peak was 15N1602-, as demonstrated by the addition of 20 sitivity. A concentration of35 mM 15N02- gave a visible signal mM 15N1602- (the dotted line in Fig. 2A). Incorporation ofdiox- after only a few scans in a 12-mm tube. Use ofthe smaller tube ygen into the newly produced N02- would have resulted in required an overnight accumulation. The pulse angle was ap- additional peaks representing 15N16018T- or 15N'80180- (or proximately 350 for the high resolution spectra of the N02 or both). Neither was observed within the resolution of the assay N03- regions. Acquisition times were 6.40 or 6.82 sec, re- (<4% ofthe 15N1602- peak). We conclude that the two oxygens spectively. The '5N1603- absorption in neutral media was de- of N02- were from water. fined as 0 ppm [to convert to the CH3N02 scale, add 3.5 ppm To further demonstrate that water was the source of oxygen (8)]. in nitrite, incubation of cells was carried out in a mixture of Materials. The '5N-labeled hydroxylamine, ammonium sul- H2160 and H2180 (60:40). Ifall oxygen ofnitrite originated from fate, and potassium nitrite were 99% enriched. Sodium nitrite H20, we predict formation of N1602-, N'60180-, and N1802- was 99% in 15N and contained little or no sodium nitrate. The with a distribution of peak intensities of 0.36, 0.48, and 0.16, H2180 and 1802 were 99% 180-enriched. respectively. These values are essentially identical to the mea- The 1802 (gas), as analyzed by gas chromatography/mass sured values of 0.35, 0.48, and 0.17, respectively, in a sample spectroscopy, contained <1% 1602. All 15N- and 180-containing of 30 mM nitrite formed by Nitrosomonas with 1602 as oxygen chemicals were from Stohler Isotopes (Waltham, MA). A 1-g source (Fig. 2B). sample-of 99% H2180 and some 1802 (gas) were supplied by Incubation ofcells with '5NH216OH and, 100% 1802 (gas) also Prochem (Summit, NJ). All other chemicals were analytical produced a single 15N'602 peak. Thus, nitrite produced from grade. Glass-distilled water was used. the intermediate hydroxylamine contained oxygen derivedfrom H20 rather than 02. Oxidation by cells of 30 umol of NH20H RESULTS in 40% H2180 and 1602 (gas) with production of 20 j.mol (33 mM) of nitrite gave a similar distribution pattern of N1602-, Size of the 180 Isotope Effect on 'N02- and N5N03-. Fig. N160180-, and N'802 (0.37, 0.45, and 0.18, respectively) as? 1A shows '5N02- signals for the three 180/16o combinations. found with ammonia.oxidation (Fig. 2C). This ratio was infor- This sample of 100 mM nitrite at pH 7.5 was made by first ex- mative. One should expect only two peaks with intensities of Downloaded by guest on September 30, 2021 Biochemistry: Andersson et d Proc. Natl. Acad. Sci. USA 79 (1982) 5873

233.6 233.4 233.2 233.0 232.8 233.4 233.2 233.0 232.8 233.4 233.2 233.0 232.8 Chemical shift, ppm FIG. 2. Nitrite formed from ammonia or hydroxylamine by Nitrosomonas has oxygens from water. (A) Formation of 40 mM [15N]nitrite (solid line) in 2 hr from 0.1 M 15NH4' and 100% 1802 in 0.25 M phosphate/5 mM carbonate solution, pH 7.0. The dotted line is the same sample containing an additional 20 mM Na'5N1602; 300 scans, 12-mm tube. (B) Formation of 30 mM ['5N]nitrite in 1.5 hr from 0.1 M 15NH4+, phosphate solution/ 100% 1602/40% H2180, pH 7.2; 812 scans, 12-mm tube. (C) Formation of 33 mM [15N]nitrite in 2 hr from 15NH20H (see Methods) in 40% H218O phosphate buffer. Final pH = 7.0; 9,240 scans, 5-mm tube (0.5 ml). 0.60 and 0.40 for N1602- and N'60180-, respectively, ifthe 160 when the starting [15N]nitrite contained a high proportion of of hydroxylamine remained in the newly formed nitrite. The 15N'802-. During cellular production of 30 mM '4N02- from presence of 0.18 N1802- shows that both oxygen atoms in ni- 1602 (by continuous oxygen bubbling) and 100 mM 14NH3 in a trite came from water. 45-min incubation, 40 mM of 15N'802- was completely con- The results indicate clearly that in oxidation of either NH3 verted to 15N1602-. Thus, again, both nitrite oxygens ex- or NH20H by cells of Nitrosomonas both atoms ofoxygen arose changed with water. from H20. Although nitrite-water exchange required concomitant oxi- Nitrosomonas Catalyzes Exchange ofthe Oxygens of Nitrite dation of ammonia or hydroxylamine, the amount of oxygen and Water During Ammonia Oxidation. The observation of exchanged could greatly exceed the amount ofnitrite produced water-derived oxygen in nitrite produced by ammonia oxidation (and ammonia oxidized) by cells during the exchange. Incu- either contradicts the conclusion that NH2OH oxygen is from bation of 12 mM 15N1602- in 40% H2180 with cells, 4 mM dioxygen (4, 6) or suggests an exchange reaction. In fact, the 14NH4 , and 1602 (gas) for 20 min caused all nitrite to acquire present results (Fig. 2C) show that the oxygen ofhydroxylamine essentially the 180 composition ofwater (Fig. 3D). This exper- exchanged with oxygens of water when hydroxylamine is oxi- iment also illustrates that the exchange of the two oxygens is a dized to nitrite by Nitrosomonas. Rees and Nason (5) observed rapid process that occurred at nitrite and ammonia concentra- a 23% exchange of the oxygens of nitrite and water during a 5- tions encountered by Nitrosomonas during culture growth. The hr incubation with cells. That slow exchange did not account rapid-exchange reaction does not occur during incubation of for the low yield of N'60180- during oxidation of NH3 in the nitrite and hydroxylamine or ammonium in the absence ofcells. presence of 1802 observed earlier (5) or in the present work. Incorporation of Oxygen from Dioxygen Is Observed when Nor can it account for the relatively rapid exchange reported Ammonia Oxidation Takes Place in the Presence of a Great here during hydroxylamine oxidation. Excess of Nitrite. As shown above, during oxidation of am- We confirm that significant nitrite exchange does not occur monia Nitrosomonas catalyzes the exchange ofoxygen between in the presence ofcells, dioxygen, and 5NO2 ; 2-hr incubation nitrite and water. Before the exchange reaction, oxygens of bio- of50 mM N'60180- with cells and 100% 1602 (gas) resulted in logically generated nitrite may, in fact, have originated from 02 no significant change in the '80- composition of nitrite (Fig. or H20. Experimentally, the origin ofoxygen remains obscured 3A and B). In remarkable contrast, after the addition of100 mM by the exchange reaction. If '5NO2- (possibly containing 180 '4NH3 and subsequent production of 25 mM 14N02-, the from dioxygen) produced by biological oxidation of [15N]ammonia 180:160 ratio of the 50 mM '5N02- (starting) nitrite had de- equilibrates with exogenously added 14N02- prior to the ex- creased from =1.0 to 0 (Fig. 3C). The same result was obtained change reaction, addition of great excess of exogenous 14N02- Downloaded by guest on September 30, 2021 5874 Biochemistry: Andersson et al. Proc. Natl. Acad. Sci. USA 79 (1982)

233.2 233.0 232.8 233.2 233.0 232.8 Chemical shift, ppm 233.4 233.2 233.0 232.8 232.6 Chemical shift, ppm FIG. 3. Nitrosomonas catalyzes an exchange of oxygens between nitrite and water. (A) Solution of 300 mM Na15N'60180 in 0.25 Mphos- FIG. 4. Incorporation of 20% of one oxygen into nitrite by Nitro- phate; 100 scans, 12-mm tube. (B) Same solution as in A diluted to 50 somonas in the presence of 100 mM exogenous nitrite. Formation of mM nitrite with phosphate buffer at pH 7.5 and incubated 2 hr with 15 mM [15Nlnitrite in 1 hr from 50 mM 15NH4' and 100% 1802 in 0.1 Nitrosomonas in 100% 1602; 180 scans, 12-mm tube. (C) Same solution M [14Nlnitrite/0.25 M phosphate/S mM carbonate solution, pH. 7.7; as inB with added 0.1 M 14NH4' incubated for 2 hr withNitrosomonas 1,760 scans, 12-mm tube. and 100% 1602; 72 scans, 12-mm tube. (D) Formation of 12 mM [15Nlnitrite after 20-min incubation of Nitrosomonas with 12 mM and to confirm the presence ofan exchange reaction associated Na15Nl602/4 mM 14NH4/100% 1602/40% H2180 in phosphate solution with oxidation ofhydroxylamine, we attempted to measure 1802 at pH 8; 7,750 scans, 12-mm tube. incorporation into 15NO2- in the presence of 15NH2OH and high 14NO2-. This experiment proved to be impossible because during oxidation of '5NH3 by bacteria should reduce the ex- of the rapid nonbiological reaction of NH20H and HNO2 to change with water of the newly produced ['5N]nitrite. This form NO and N20. would allow identification of the oxygen source. With this in mind, cells were incubated with 50 mM 15NH4+, DISCUSSION 50% 1802 (gas)/50% Ar, and varying concentrations of 14NO2-. Use of '5N (180) NMR in Biology. The present report de- Incubation with 50 (1 hr), 100 (1 hr), or 150 (2 hr) mM 14NO2- scribes the use of the 180 isotope shift 15N NMR in the study allowed production of 15%, 20%, or 9%, respectively, of of biological transformation of N oxides. The principle is the ['5N]nitrite as the N'60180- form. The same result was ob- same as with 31p (180) NMR, which alsohas been used in biology tained with 100 mM 14NO2- and 100% 1802 (gas) (Fig. 4). A peak (14). We note that the separation ofthe nitrite peaks in 15N (180) associated with '5N1802 was not detected in this experiment, NMR (0.13 ppm, or 4.2 Hz at 7.05 T) is greater than for phos- possibly because the expected quantity (=4%) is close to the phate in 31p ('80) NMR (0.02 ppm, or 2.4 Hz at 7.05 T). The limit of measurement. We note that these are unusually high peak separation in nitrite and nitrate reported here agrees well concentrations ofnitrite and that the rate ofammonia oxidation with published values (9). We believe that the technique has was, in fact, progressively inhibited (20%, 40%, or 50% inhi- great potential: (i) it permits clear differentiation between 160 bition at 50, 100, or 150 mM nitrite, respectively). The results and 180 derivatives of '5N by simply measuring peak heights are consistent with the conclusion of Rees and Nason (5) that and permits better resolution than with 170 as in 31p (170) NMR at least one nitrite oxygen originates from 02. (15); (ii) the compound of interest may be enriched with 15N, We note that the 15N160'80- produced in these experiments thus eliminating background 15N signals from the biological (Fig. 4) is stable in the presence of H216o. Thus, factors nec- mixture; (iii) measurement can be made directly on soluble essary for the N02--H20 exchange reaction are not present in compounds in the reaction solution. The latter aspect eliminates the supernatant that results from sedimentation of cells from the necessity for chemical conversion of soluble compounds to the reaction mixture. gaseous products. The technique is illustrated here with nitrite To determine the origin ofoxygen in NH2OH-derived nitrite and nitrate. Downloaded by guest on September 30, 2021 Biochemistry: Andersson et al. Proc. Natl. Acad. Sci. USA 79 (1982) 5875 We suggest that the approach will prove useful in the study Identification of Source of Oxygen in Nitrite. The work of of microbiological oxidation and reduction of N oxides as well Hollocher et aL (6) clearly indicates that ammonia is oxygenated as the synthesis of organic N oxides and hydroxamates by mi- (from 02) in the formation of hydroxylamine by Nitrosomonas. croorganisms and higher organisms (e.g., aryl N oxides or N Preliminary work by our group indicates that oxidation of hy- hydroxy-products of liver microsomal cytochrome P450 me- droxylamine to nitrite by purified hydroxylamine oxidoreduc- tabolism). The straightforward interpretation of this '5N NMR tase also involves the incorporation of oxygen from 02 (unpub- technique makes it readily accessible. lished). Taken together, these observations seem to indicate Exchange ofOxygen Between H20 and HNO2. Demonstra- that both oxygens of nitrite produced from NH3 in vivo by Ni- tion of the exchange with water of both oxygens of nitrite as trosomonas originate from 02. However, the latter conclusion catalyzed by Nitrosomonas is significant because it explains the depends on the assumptions that (i) hydroxylamine is, in fact, previously observed difficulty in assessing the source ofoxygen an intermediate in vivo in oxidation of ammonia to nitrite; (ii) in nitrite produced from ammonia or hydroxylamine. Further, oxidation of hydroxylamine does not involve the initial removal it may provide useful clues to the mechanism of oxidation of of oxygen-i.e., dehydration of enzyme-bound substrate ammonia andhydroxylamine. The present work establishes sev- (NH2OH -k [NH] + H20; [NH] +2 - HNO2); and (iii) the eral aspects of the exchange: (i) The exchange reported here in vitro oxygen addition reaction of hydroxylamine oxidore- differs from the acid-catalyzed nitrite-water exchange observed ductase is the same as that in vivo (even though very significant at pH 6.26 (9) in that the rate at neutral pH is at least 2 orders differences such as production ofnitrate have been noted; ref. ofmagnitude greater and both oxygens are exchanged. (ii) It is 18). dependent on the simultaneous oxidation of ammonia or hy- Elimination of these uncertainties requires direct demon- droxylamine to nitrite but the amount of nitrite exchanged is stration of cell-catalyzed 1802 incorporation into nitrite with not stoichiometric with the amount of ammonia or hydroxyl- wNH3 as substrate. By using '5N NMR, the present-work has amine oxidized or nitrite produced. For example, we have ob- shown that at least 20% of the oxygens in NH3-derived nitrite served that the exchange of 1 mole of nitrite requires the oxi- originate from dioxygen. This is the best evidence to date that dation of no more than 0.33 mole of ammonia. This result at least one ofthe oxygens ofnitrite produced during ammonia excludes a stoichiometry ofone or two oxygens exchanged per oxidation does, in fact, originate from 02 NH3 oxidized to N02-. (iii) During oxidation of NH2OH to ni- trite, the oxygen of hydroxylamine is exchanged with H20. We thank Ms. Celine Lyman for growing the bacteria and Dr. J. Therefore, the exchange is presumed to occur in a N compound Lipscomb for stimulating discussions. This research was supported by subsequent to hydroxylamine in the pathway, possibly nitrite Grant PCM 8008710 to A.B.H. from the National Science Foundation. itself. (iv) Biologically produced nitrite apparently equilibrates 1. Hooper, A. B. (1978) in Microbiology, ed. Schlessinger, D. (Am. with exogenously added nitrite. This allowed saturation of the Soc. Microbiol., Washington, DC), pp. 299-304. exchange mechanism with 2. Lipschultz, F., Zafiriou, 0. C., Wofsg, S. C., McElroy, M. G., 14NO2 and incorporation of up to Valois, F. W. & Watson, S. W. (1981) Nature (London) 294, 641. 20% of one of the two oxygens of '5NO2 derived from dioxy- 3. Lees, H. (1952) Nature (London) 169, 156. gen during biological oxidation of '5NH3. 4. Dua, R. D., Bhandari, B. & Nicholas, D. J. D. (1979) FEBS Lett. The data do not allow description of the mechanism of the 196, 401-404. exchange reaction, but several possibilities deserve mention. 5. Rees, M. & Nason, A. (1965) Biochim. Biophys. Acta 113, 398- It maybe that biological oxidation ofammonia or hydroxylamine 401. involves 6. Hollocher, T. C., Tate, M. E. & Nicholas, D. J. D. (1981)J. Bio. reaction ofan intermediate ofnitrogen or oxygen with Chein. 256, 10834-10836. nitrite to form a second compound (possibly also an interme- 7. Levy, G. C. & Lichter, R. L. (1979) Nitrogen-15 NMR Spectros- diate in the pathway) containing H20-exchangeable oxygens. copy (Wiley, New York). The formation of the hypothetical second compound must be 8. Martin, G. J., Martin, M. L. & Gouesnard, J.-P. (1981)15N NMR rapid and reversible to account for the high value of nitrite ex- Spectroscopy (Springer, Berlin). changed per ammonia oxidized. In light of this possibility, an 9. Van Etten, R. L. & Risley, J. M. (1981) J. Am. Chem. Soc. 103, investigation by NMR of a possible intermediate 5633-5636. N-containing 10. Hooper, A. B., Maxwell, P. C. & Terry, K. R. (1978) Biochem- in the exchange reaction should be undertaken. istry 17, 2984-2989. An alternative explanation is that the nitrite-water oxygen 11. Nicholas, D. J. D. & Nason, A. (1957) Methods Enzymol 3, 981- exchange reaction may be dependent upon, but not a direct part 984. of, the NH3 or NH2OH oxidative pathway. For example, a re- 12. Anbar, M., Halman, N. & Pinchas, S. J. (1960) J. Chem. Soc., gion ofhigh acidity (pH = 4) generated by NH2OH-dependent 1242-1245. proton pumping might promote chemical exchange of 13. Jordan, S. & Bonner, F. T. (1973) Inorg. Chem. 12, 1369-1374. nitrite 14. Cohn, M. & Hu, A. (1978) Proc. Natl. Acad. Sci. USA 75, 200- oxygens with water (the same reaction used to chemically syn- 203. thesize '80-enriched nitrite). The hypothesized region of low 15. Tsai, M. D., Huang, S. L., Kozlowski, J. F. & Chang, G. C. pH would be "extracellular" and possible in the lumen of the (1980) Biochemistry 19, 3531-3536. unique intracellular membrane system of Nitrosomonas. Fi- 16. Hooper, A. B. (1968) Biochim. Biophys. Acta 162, 49-65. 17. Erickson, R. H., Hooper, A. B. & Terry, K. R. (1972) Biochim. nally, exchange of nitrite oxygen as part of other reactions not Biophys. Acta 283, 155-166. linked directly to ammonia oxidation cannot be excluded-e.g., 18. Hooper, A. B., Terry, K. R. & Maxwell, P. C. (1977) Biochim. the nitrite reductase (16) or terminal oxidase (17) ofNitrosomonas. Biophys. Acta 462, 141-152. Downloaded by guest on September 30, 2021