Nitrogen and oxygen isotopic fractionation during microbial nitrite reduction

Microbial nitrite reduction plays an important role in the nitrogen cycle, producing the first gaseous product in the denitrification pathway. The role of nitrite reduction in the environment can be assessed using stable isotope measurements of nitrite. Here, we present estimates for nitrogen (N) and oxygen (O) isotope fractionation during nitrite reduction catalyzed by copper-containing nitrite reductase (Cu-NIR) and cytochrome cd1-containing nitrite reductase (Fe-NIR). A Rayleigh fractionation model was used to calculate the N and O isotope effects, (15)epsilon and (18)epsilon respectively, from time-course measurements of nitrite concentration and isotopic composition in batch culture experiments. For three strains of denitrifier carrying the Cu-NIR, (15)epsilon=22 +/- 2 parts per thousand and (18)epsilon=2 +/- 2 parts per thousand (95% confidence interval). For three strains of denitrifier carrying the Fe-NIR, (15)epsilon=8 +/- 2 and (18)epsilon=6 +/- 2 parts per thousand (95% confidence interval). These isotope effects for nitrite reduction are significantly different from each other. Furthermore, (15)epsilon and (18)epsilon do not show a 1 : 1 relationship, as has been assumed. The difference between the isotope effects for these two families of enzymes is likely due to a mechanical difference in how the enzymes bind nitrite. The Cu-NIR binds to both O atoms and the Fe-NIR only binds to the N, allowing either N-O bond to be cleaved and imparting a larger isotope effect for O than for the Cu-NIR. Utilizing these new N isotope effects for nitrite reduction in oxygen minimum zone N cycle models results in higher rates of nitrite oxidation than previously modeled.