Enzyme level N and O isotope effects of assimilatory and dissimilatory nitrate reduction
To provide mechanistic constraints to interpret nitrogen (N) and oxygen (O) isotope ratios of nitrate ( NO 3 − ), 15N/14N and 18O/16O, in the environment, we measured the enzymatic NO 3 − N and O isotope effects (15 ε and 18 ε) during its reduction by NO 3 − reductase enzymes, including (1) a prokar...
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Veröffentlicht in: | Limnology and oceanography 2017-01, Vol.62 (1), p.272-288 |
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Sprache: | eng |
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Zusammenfassung: | To provide mechanistic constraints to interpret nitrogen (N) and oxygen (O) isotope ratios of nitrate (
NO
3
−
), 15N/14N and 18O/16O, in the environment, we measured the enzymatic
NO
3
−
N and O isotope effects (15
ε and 18
ε) during its reduction by
NO
3
−
reductase enzymes, including (1) a prokaryotic respiratory
NO
3
−
reductase, Nar, from the heterotrophic denitrifier Paracoccus denitrificans, (2) eukaryotic assimilatory
NO
3
−
reductases, eukNR, from Pichia angusta and from Arabidopsis thaliana, and (3) a prokaryotic periplasmic
NO
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 during [
NO
3
−
] assimilation and denitrification, reinforcing the reliability of this benchmark to identify
NO
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. |
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ISSN: | 0024-3590 1939-5590 |
DOI: | 10.1002/lno.10393 |