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.