Dataset: N and O isotope effects of nitrate reductases

Sources of Nitrate Reductase:
Enzymatic assays were conducted on (a) cell homogenates from the denitrifying bacterial strain Paracoccus denitrificans (American Type Culture Collection [ATCC] 19367) cultured under anaerobic vs. aerobic conditions, on (b) cell homogenates from the photo-heterotrophic bacterial strain Rhodobacter sphaeroides (Deutsche Sammlung von Mikroorganismen [DSM] 158) cultured aerobically, and on (c) purified extracts of recombinant eukaryotic assimilatory nitrate reductases (EukNR) from the flowering plant Arabidopsis thaliana (AtNaR:  E.C. 1.7.1.1) and from the yeast Pichia angusta (YNaR1: E.C. 1.7.1.2), both purchased from NECi (nitrate.com).

Enzymatic assay preparations:
Assays contained 0.5 or 1 mL of cell suspension or of commercially purified EukNR buffered solution, 1 mL of 200 umol L-1 reducing agent – either membrane-permeant benzyl viologen dichloride [Sigma-Aldrich, CAS: 1102-19-8], methyl viologen dichloride hydrate [Sigma-Aldrich, CAS: 75365-73-0], or hydroquinone (for dissimilatory reductases only; [MP Organics]) –  0.2 or 1 mL 10 mmol L-1 KNO3 to a final concentration of 200 or 1000 umol L-1, and the remaining volume of 100 mmol L-1 phosphate buffer [pH 7.9] containing 100 umol L-1 to a final assay volume of 10 mL. After drawing an initial 1 mL sample for quantitation of [NO3-] and [NO2-], the denitrification reaction was commenced by the addition of 1 mL of 57 mmol L-1 sodium dithionite in 29 mmol L-1  sodium bicarbonate, which reduces the electron donor. Initial [NO3-] and [NO2-] values are corrected for this dilution. Serial 1 mL samples were drawn approximately every 90 seconds during room temperature assays and every 3 minutes during assays conducted at 4 degrees C. Samples were mixed vigorously on a vortex mixer for 30 s immediately upon collection to halt the reaction through oxidation of the methyl or benzyl viologen or hydroquinone.  In selected assays, additional ~ 50 uL samples were also drawn throughout the assay reactions for determination of [NO2-] and were measured immediately. In order to ensure complete cessation of enzyme activity, samples placed in an 80 degrees C water bath for 2 to 10 minutes. NO2- was then removed from the samples via the addition of 55 uL 4% (wt/vol) sulfamic acid in 10% vol/vol HCl.

Determination of [NO3-]:
[NO3-] was measured in samples by chemiluminescence detection on a NOx analyzer (model T200 Teledyne Advanced Pollution Instrumentation) following reduction to NO in a heated vanadium solution (Braman and Hendrix 1989). Nitrite had been previously removed from these samples, as vanadium reduces both nitrate and nitrite to NO.

Nitrate N and O isotope ratio analyses:
NO3- δ15N and δ18O were determined with the denitrifier method (Sigman et al. 2001; Casciotti et al. 2002), wherein denitrifying bacteria lacking terminal nitrous oxide reductase (P. chlororaphis f. sp. aureofaciens ATCC 1398) quantitatively convert sample NO3- to N2O gas, which is then extracted, purified and analyzed through a modified Thermo-Scientific Gas Bench II and Delta V Advantage gas chromatograph isotope ratio mass spectrometer.  Samples were standardized through comparison to reference standards IAEA-N3, USGS-34, and USGS-32, which have δ15N (vs air N2) and δ18O (vs V-SMOW) of 4.7‰ and 25.6‰, -1.8‰ and -27.9‰, and 180‰ and 25.6‰ respectively after individually being referenced to pure N2O injections from a common reference gas cylinder.