18 O isotope shift in 15 N NMR analysis of biological N-oxidations: H 2 O-NO 2 - exchange in the ammonia-oxidizing bacterium Nitrosomonas

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Abstract
The 18O/16O shifts in 15N NMR were determined for nitrite (0.13 ppm or 4.2 Hz at 7.05 T) and nitrate (0.056 ppm or 1.7 Hz at 7.05 T) at neutral pH. The technique, which allows clear differentiation between 16O and 18O derivatives of 15N, was used to assess the source of oxygens in nitrite produced by oxidation of ammonia in N. europaea. The 2 oxygens of nitrite produced by cell-catalyzed oxidation of ammonia or hydroxylamine had the 16O/18O isotope composition of water. Nitrosomonas catalyzed the rapid exchange of O2 between nitrite and water. The exchange reaction required the concomitant oxidation of ammonia. The amount of nitrite exchanged could exceed the amount of ammonia oxidized by a factor of 3. This exchange explains previous difficulties in the determination of the source of nitrite oxygen in ammonia oxidation. When cells oxidized [15N] ammonia in the presence of a great excess of exogenous [14N]nitrite, 20% of oxygen in the resulting [15N]nitrite was derived from dioxygen. Dioxygen is apparently the source of at least 1 oxygen in nitrite produced by Nitrosomonas.