Enzyme level N and O isotope effects of assimilatory and dissimilatory nitrate reduction. (10th October 2016)

• Record Type: Journal Article
• Authors: Treibergs, Lija A.; Granger, Julie
• Is Part Of: Limnology and oceanography. Volume 62:Number 1(2017)
• Page Start: 272
• Page End: 288
• Publication Date: 2016-10-10
• Subjects: Limnology -- Periodicals; Oceanography -- Periodicals; Océanographie; Limnologie; Limnology; Oceanography; Computer network resources; Périodique électronique (Descripteur de forme); Ressource Internet (Descripteur de forme); Periodicals; 551.4805
• DOI: 10.1002/lno.10393
• Languages: English
• ISSNs: 0024-3590

Abstract

Abstract: 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 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.