Abstract. Nitrous oxide (N2O) is a potent greenhouse gas and produced in denitrification and nitrification by various microorganisms. Site preference (SP) of 15N in N2O, which is defined as the difference in the natural abundance of isotopomers 14N15NO and 15N14NO relative to 14N14NO, 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 O2 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.

Tetraheme cytochrome c-554 is a physiological electron acceptor of hydroxylamine oxidoreductase (HAO), a core enzyme of ammonia oxidation in chemoautotrophic nitrifiers. Here we report the purification of cytochrome c-554 from Nitrosococcus oceani strain NS58, a marine gammaproteobacterial ammonia-oxidizing bacterium. The NS58 cytochrome is a 25 kDa-protein having four hemes c. The absorption spectrum of the cytochrome showed peaks at 420 nm, 523 nm, and 554 nm, with shoulders at around 430 nm and 580 nm in the reduced state. In contrast to the highly basic counterpart from the betaproteobacterium Nitrosomonas europaea, the NS58 cytochrome c-554 was an acidic protein whose isoelectric point was 4.6. HAO was also purified, and the reaction with the NS58 cytochrome was found to be salt-tolerant. Compared with the activity observed in a non-salt solution, 60% of the activity remained in a saline concentration comparable to that of seawater.

Nitrous oxide (N₂O) is a potent greenhouse gas and produced in denitrification and nitrification in environmental nitrogen cycle by various microorganism. Site preference (SP) of ¹⁵N in N₂O, which is defined as the difference in the natural abundance of isotopomers ¹⁴N¹⁵NO and ¹⁵N¹⁴NO relative to ¹⁴N¹⁴NO, has been reported to be a useful tool to quantitatively distinguish N₂O production pathway. To determine representative SP value 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, respectively. 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 O₂ availability. Our calculations revealed that previous in vivo studies might have underestimated the SP value for 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

Volume 25, no. 2, Page 95-102, 2010 Page 98, column 2, line 28 from the top, 'The N-terminal amino acid sequence of the protein was determined as IPDELYEALGVDKYKASPKE. The sequence was identical to the deduced sequence of the 31st to 50th residues of the HAO precursor encoded in the noc_0892 gene of the N. oceani ATCC19707' should read 'The N-terminal amino acid sequence of the protein was determined as DIPDELYEALGVDKYKASPK. The sequence was identical to the deduced sequence of the 30th to 49th residues of the HAO precursor encoded in the noc_0892 gene of the N. oceani ATCC19707'.