15N tracer studies on the reduction of nitrite by the purified dissimilatory nitrite reductase of Pseudomonas aeruginosa. Evidence for direct production of N2O without free NO as an intermediate.

Nitrite reductase (cytochrome c,d1) was purified from Pseudomonas aeruginosa. In the presence of the reducing system, ascorbate-N,N,N',N'-tetramethylphenyl-enediamine, which alone had no ability to reduce nitrite or NO at pH 7.5, the enzyme catalyzed the reduction of nitrite to NO and N2O as major and minor products, respectively, as determined by gas chromatography-mass spectrometry. The rate of reduction of NO to N2O was considerably lower than the rate of reduction of nitrite to N2O and might be zero. The N2O produced in a system containing [15N]nitrite and natural NO was more highly enriched in 15N than was the NO pool and, in this regard, closely resembled the enrichment of the nitrite pool. The amount of 14N in the NO pool changed little, if any, as the result of enzymatic processes. For the enzyme, free NO seems not to be an intermediate between nitrite and N2O, just as was found by this laboratory for certain intact denitrifying bacteria. The results are consistent with reduction of nitrite to enzyme-bound NO, which can partition between release and further reduction.