Isotopomeric characterization of nitrous oxide produced by reaction of enzymes extracted from nitrifying and denitrifying bacteria

Nitrous oxide (N2O) is a potent greenhouse gas and produced in denitrification and nitrification by various microorganisms. Site preference (SP) of N-15 in N2O, which is defined as the difference in the natural abundance of isotopomers (NNO)-N-14-N-15 and (NNO)-N-15-N-14 relative to (NNO)-N-14-N-14, has been reported to be a useful tool to quantitatively distinguish N2O production pathways. To determine representative SP values for each microbial process, we firstly measured SP of N2O produced in the enzyme reaction of hydroxylamine oxidoreductase (HAO) purified from two species of ammonia oxidizing bacteria (AOB), Nitrosomonas europaea and Nitrosococcus oceani, and that of nitric oxide reductase (NOR) from Paracoccus denitrificans. The SP value for NOR reaction (-5.9 +/- 2.1 %) showed nearly the same value as that reported for N2O produced by P. denitrificans in pure culture. In contrast, SP value for HAO reaction (36.3 +/- 2.3 %) was a little higher than the values reported for N2O produced by AOB in aerobic pure culture. Using the SP values obtained by HAO and NOR reactions, we calculated relative contribution of the nitrite (NO2-) reduction (which is followed by NO reduction) to N2O production by N. oceani incubated under different O-2 availability. Our calculations revealed that previous in vivo studies might have underestimated the SP value for the NH2OH oxidation pathway possibly due to a small contribution of NO2- reduction pathway. Further evaluation of isotopomer signatures of N2O using common enzymes of other processes related to N2O would improve the isotopomer analysis of N2O in various environments.