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Enzyme level N and O isotope effects of assimilatory and dissimilatory nitrate reduction
Author(s) -
Treibergs Lija A.,
Granger Julie
Publication year - 2017
Publication title -
limnology and oceanography
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.7
H-Index - 197
eISSN - 1939-5590
pISSN - 0024-3590
DOI - 10.1002/lno.10393
Subject(s) - chemistry , nitrate reductase , paracoccus denitrificans , biochemistry , enzyme , reductase , rhodobacter sphaeroides , photosynthesis
To provide mechanistic constraints to interpret nitrogen (N) and oxygen (O) isotope ratios of nitrate (NO 3 −), 15 N/ 14 N and 18 O/ 16 O, in the environment, we measured the enzymaticNO 3 −N and O isotope effects ( 15 ε and 18 ε) during its reduction byNO 3 −reductase enzymes, including (1) a prokaryotic respiratoryNO 3 −reductase, Nar , from the heterotrophic denitrifier Paracoccus denitrificans , (2) eukaryotic assimilatoryNO 3 −reductases, eukNR , from Pichia angusta and from Arabidopsis thaliana , and (3) a prokaryotic periplasmicNO 3 −reductase, Nap , from the photoheterotroph Rhodobacter sphaeroides . Enzymatic Nar and eukNR assays with artificial viologen electron donors yielded identical 18 ε and 15 ε of ∼28‰, regardless of [NO 3 −] or assay temperature, suggesting analogous kinetic mechanisms with viologen reductants. Nar assays fuelled with the physiological reductant hydroquinone (HQ) also yielded 18 ε ≈ 15 ε, but variable amplitudes from 21‰ to 33.0‰ in association with [NO 3 −], suggesting analogous substrate sensitivity in vivo. Nap assays fuelled by viologen revealed 18 ε: 15 ε of 0.50, where 18 ε ≈ 19‰ and 15 ε ≈ 38‰, indicating a distinct catalytic mechanism than Nar and eukNR . Nap isotope effects measured in vivo showed a similar 18 ε: 15 ε of 0.57, but reduced 18 ε ≈ 11‰ and 15 ε ≈ 19‰. Together, the results confirm identical enzymatic 18 ε and 15 ε duringNO 3 −assimilation and denitrification, reinforcing the reliability of this benchmark to identifyNO 3 −consumption in the environment. However, the amplitude of enzymatic isotope effects is apt to vary in vivo. The distinctive signature of Nap is of interest for deciphering catalytic mechanisms but may be negligible in most environments given its physiological role.