Isotope labeling studies on the mechanism of N-N bond formation in denitrification

The mechanism of the denitrification and nitrosation reactions catalyzed by the heme cd-containing nitrite reductase from Pseudomonas stutzeri JM 300 has been studied with whole cell suspensions using H2(18)O, 15NO, and 15NO-2. The extent of H2(18)O exchange with the enzyme-bound nitrosyl intermediate, as determined by the 18O content of product N2O, decreased with increasing nitrite concentration, which is consistent with production of N2O by sequential reaction of two nitrite ions with the enzyme. Reaction of NO with whole cells in H2(18)O gave amounts of 18O in the N2O product consistent with equilibration of nitric oxide with a small pool of free nitrite. Using 15NO and NH2OH, competition between denitrification and nitrosation reactions was demonstrated, as is required if the enzyme-nitrosyl complex is an intermediate in both nitrosation and denitrification reactions. The first evidence for exchange of 18O between H2(18)O and a nitrosation intermediate occurring after the enzyme-nitrosyl complex, presumably an enzyme-bound nitrosamine, has been obtained. The collective results are most consistent with denitrification N2O originating via attack of NO-2 on a coordinated nitrosyl, as proposed earlier (Averill, B. A., and Tiedje, J. M. (1982) FEBS Lett. 138, 8-11).