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

The (18)O/(16)O shifts in (15)N 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 (16)O and (18)O derivatives of (15)N, was used to assess the source of oxygens in nitrite produced by oxidation of ammonia in Nitrosomonas. The two oxygens of nitrite produced by cell-catalyzed oxidation of ammonia or hydroxylamine had the (16)O/(18)O isotope composition of water. Nitrosomonas is shown to catalyze the rapid exchange of oxygen 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 [(15)N]ammonia in the presence of a great excess of exogenous [(14)N]nitrite, 20% of one oxygen in the resulting [(15)N]nitrite was derived from dioxygen. Dioxygen is apparently the source of at least one oxygen in nitrite produced by Nitrosomonas.