Nitrogen 15 tracer studies on the pathway of denitrification in Pseudomonas aeruginosa

The pathway of anaerobic reduction of nitrite to nitrogen gas (N2) by cell suspensions of the denitrifier, Pseudomonas aeruginosa, was studied using the techniques of gas chromatography and mass spectrometry. While release of nitrous oxide (N2O) is not normally detected during the reduction of nitrite to N2 by this organism, 15N from [15N]nitrite nevertheless can be trapped quantitatively as 15N2O in a pool of added N2O. In such experiments the abundance of 15N in N2O always exceeds that in product N2, consistent with the absence of a major reductive route from nitrite to N2 which by-passes N2O. During the reduction of a mixture of [15N]nitrite and nitric oxide (NO), 15NO produced at most only in trace amounts. The final products are chiefly 15N2 and 14N2 with only a small fraction of the scrambled product, 14N15N. Much of the 14N15N can be accounted for as an artifact caused by traces of molecular oxygen, which promote the conversion of NO to nitrite by autooxidation and thereby degrade slightly the isotopic purity of [15N]nitrite. Nitrous oxide shows all the properties of a free obligatory intermediate during the denitrification of nitrite to N2 by P. aeruginosa, whereas NO does not. The inability to trap 15NO in a pool of NO indicates that NO is not a free obligatory intermediate in the reduction of nitrite. The small mole fractions of 14N15N produced from a mixture of [15N]nitrite and NO require that the main reductive pathways for these nitrogen oxides cannot share any freely diffusible mono-nitrogen intermediate in common. The simplest interpretation is that nitrite and NO are denitrified by separate pathways, at least prior to the formation of the first bi-nitrogen compound.