Eukaryotic Assimilatory Nitrate Reductase Fractionates N and O

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Eukaryotic Assimilatory Nitrate Reductase Fractionates N and O Isotopes with a Ratio near Unity Kristen L. Karsh,*,†,‡,§,∥ Julie Granger,†,⊥ K. Kritee,†,# and Daniel M. Sigman† †

Department of Geosciences, Princeton University, Guyot Hall, Princeton, New Jersey 08544, United States Antarctic Climate & Ecosystems Cooperative Research Centre, University of Tasmania, Private Bag 80, Hobart, Tasmania 7001 Australia § Institute for Marine and Antarctic Studies, University of Tasmania, Private Bag 129, Hobart, Tasmania 7001 Australia ∥ CSIRO Marine and Atmospheric Research, GPO Box 1538, Hobart, Tasmania 7001 Australia ‡

S Supporting Information *

ABSTRACT: In order to (i) establish the biological systematics necessary to interpret nitrogen (N) and oxygen (O) isotope ratios of nitrate (15N/14N and 18O/16O) in the environment and (ii) investigate the potential for isotopes to elucidate the mechanism of a key N cycle enzyme, we measured the nitrate N and O isotope effects (15ε and 18ε) for nitrate reduction by two assimilatory eukaryotic nitrate reductase (eukNR) enzymes. The 15ε for purified extracts of NADPH eukNR from the fungus Aspergillus niger and the 15 ε for NADH eukNR from cell homogenates of the marine diatom Thalassiosira weissf logii were indistinguishable, yielding a mean 15ε for the enzyme of 26.6 ± 0.2‰. Both forms of eukNR imparted near equivalent fractionation on N and O isotopes. The increase in 18O/16O versus the increase in 15N/14N (relative to their natural abundances) was 0.96 ± 0.01 for NADPH eukNR and 1.09 ± 0.03 for NADH eukNR. These results are the first reliable measurements of the coupled N and O isotope effects for any form of eukNR. They support the prevailing view that intracellular reduction by eukNR is the dominant step in isotope fractionation during nitrate assimilation and that it drives the 18ε:15ε ≈ 1 observed in phytoplankton cultures, suggesting that this O-to-N isotope signature will apply broadly in the environment. Our measured 15 ε and 18ε may represent the intrinsic isotope effects for eukNR-mediated N−O bond rupture, a potential constraint on the nature of the enzyme’s transition state.

1. INTRODUCTION Nitrogen is an essential component of all living organisms. Because relatively little nitrogen is in a form that can be assimilated by plants, nitrogen often limits primary production in marine and terrestrial ecosystems. It is also a limiting factor in agricultural production and, due to the use of nitrogen fertilizers to address this, anthropogenic influence on the nitrogen cycle is escalating.1,2 Thus, in many areas of research, understanding nitrogen cycling at local, regional, and global scales is a major goal. The nitrogen (N) and oxygen (O) isotope ratios of nitrate (15N/14N and 18O/16O, respectively) are in many ways ideal tools for studying the sources, transport, internal cycling, and natural inputs and losses of nitrogen. The natural abundances of the stable isotopes integrate over small scale variability to yield coherent mean rates of processes, and neither the isotope ratios nor their measurement alter the system that they are being used to study. The use of both N and O isotopes of nitrate overcomes a key limitation of using N isotopes alone, in that N isotope ratios in and of themselves © 2012 American Chemical Society

cannot separate co-occurring processes that have counteracting effects on the N isotope ratio.3 The two major nitrate consuming processes, nitrate assimilation and denitrification, cause significant isotopic fractionation of nitrate. Nitrate containing the light isotope (14N or 16O) is consumed more rapidly than nitrate containing the heavy isotope (15N or 18O), leaving the residual nitrate pool with higher 15N/14N and 18O/16O ratios. The relative rates of reaction of the heavy and light isotopes are reflected in the kinetic isotope effect ε, where ε = (lightk /heavyk −1) (reported in permil, ‰). Both nitrate assimilation and marine denitrification fractionate N and O isotopes in a ratio near 1 (i.e., 18ε:15ε ≈ 1) in laboratory cultures and in the environment.3−7 The signature 18 15 ε: ε ≈ 1 associated with nitrate assimilation and marine denitrification constitutes the cornerstone of studies using Received: Revised: Accepted: Published: 5727

December 29, 2011 April 20, 2012 April 25, 2012 April 25, 2012 dx.doi.org/10.1021/es204593q | Environ. Sci. Technol. 2012, 46, 5727−5735

Environmental Science & Technology

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of the important transformations and a better understanding of the underlying enzymatic isotope effects. Here, we begin to address this need, focusing on N and O isotopic fractionation by assimilatory nitrate reductase enzymes. In order to (i) test the hypothesis that the assimilatory eukaryotic nitrate reductases impart equivalent fractionation on N and O isotopes of nitrate, (ii) test the prevailing reductasecentered model of organism-level isotope fractionation during nitrate assimilation, and (iii) assess the potential for isotopes to elucidate the mechanism of this important enzyme, we measured nitrate N and O isotope fractionation by eukaryotic assimilatory nitrate reductase from the fungus Aspergillus niger and from the marine diatom Thalassiosira weissf logii.

coupled N and O measurements; with deviations from an O-toN ratio of ∼1 in environmental samples indicating the presence of other N cycle transformations. In particular, nitrate production via nitrification has different effects on the N and O isotopes of nitrate; the δ15N of newly produced (i.e., nitrified) nitrate depends on the origin of N being nitrified, while the δ18O of nitrate does not.3 One can derive the isotopic composition of the nitrate added by nitrification based on deviation in δ18O and δ15N from a ratio of ∼1 and in so doing, gain information on its ultimate source (e.g., nitrogen fixation or nitrate cycling within a water parcel).3 Thus, coupled N and O isotopes have been used in a broad range of applications, including (i) quantifying N2 fixation in regions of marine denitrification,3 (ii) identifying nitrification in rapidly denitrifying marine sediments,8,9 (iii) identifying the source of nitrate in the surface mixed layer of the ocean (nitrate regenerated in situ via nitrification versus “new” nitrate supplied from depth),7,10 and (iv) delineating the source, sinks, and cycling of N in watersheds, estuaries, and river basins.11−13 As yet, the association of nitrate assimilation and marine denitrification with an 18ε:15ε ≈ 1 is based on empirical evidence from cultureand field-based studies5,6,14 without full understanding of the physiological mechanisms generating the signature ratio. The current hypothesis that the observed ratio originates with equivalent fractionation of the N and O isotopes by nitrate reductase enzymes remains untested.5,6,14 Looking beyond the coupled N and O relationship of nitrate isotope fractionation, the absolute magnitude of the organismlevel N isotope effect (i.e., the net isotope effect expressed in the environment, 15εorg) is an important parameter for studies using 15N/14N distributions to constrain surface ocean nitrate supply and biological consumption in the present15,16 and past.17,18 Because the magnitude of the organism-level N isotope effect can vary, see for example, refs 6 and 19, a mechanistic understanding of N (and O) isotope fractionation during nitrate assimilation would strengthen environmental application of nitrate isotopes. Intracellular reduction by the nitrate reductase enzyme is thought to be the dominant fractionating step in nitrate assimilation; efflux of 15N-enriched nitrate to the medium expresses this fractionation outside the cell.20−23 Measuring an enzymatic isotope effect of a magnitude greater than that observed in the environment would provide strong evidence for a reductase-driven model of nitrate isotope fractionation during nitrate assimilation. Isotope fractionation associated with enzymatic reactions can also provide information about the enzyme’s kinetic mechanism and the nature of transition states.24 Nitrate reduction by eukaryotic assimilatory nitrate reductase (eukNR) is a ratelimiting step in nitrate assimilation by plants25 and algae;26 therefore, an understanding of the enzyme’s catalytic mechanism and regulation is of interest.25 Thus far, there have been few direct measurements of the N and O isotope effects for eukNR27−29 and few applications to the investigation of enzyme mechanism.30 The last reported measurements of isotope fractionation by nitrate reductase enzymes were made over 25 years ago and yielded conflicting results [15ε = 15‰,27 and 15ε = 30‰ and 18ε = 15‰28,29 for assimilatory eukaryotic nitrate reductase (eukNR), see Table S1 of the SI]. The methods used to measure variation in the N and O isotope ratios of nitrate have since dramatically improved.4,31,32 For the N and O isotopes of nitrate to reach their full potential as a biogeochemical tracer, we require better estimates of the organism-level isotope effects

2. MATERIALS AND METHODS 2.1. Sources of Nitrate Reductase. Assays were conducted on (i) purified extracts of NADPH eukaryotic assimilatory nitrate reductase (eukNR) from A. niger (EC 1.7.1.2, formerly EC 1.6.6.2, purchased from Sigma-Aldrich, USA) and on (ii) NADH eukNR (EC 1.7.1.1, formerly EC 1.6.6.1) in cell homogenates from the marine diatom T. weissf logii following extraction from cell cultures. Culture conditions for T. weissf logii and procedures for sampling and enzyme extraction are described in supporting text S2.1 of the Supporting Information, SI. NADPH eukNR from A. niger was chosen because sufficient amounts of purified enzyme could be obtained readily (and therefore the majority of experimentation was on this form of the enzyme). T. weissf logii assays were conducted (i) to compare isotopic fractionation by the NADH form of the enzyme with that of the NADPH form and (ii) for direct comparison with predictions of the eukNR isotope effect based on T. weissf logii cell cultures.22,33 The NADPH-specific and NADH-specific forms of eukNR span the breadth of diversity in the monophyletic eukNR enzyme family,34 such that similar behavior from the two forms would argue for uniformity across eukNR enzymes in general. 2.2. Enzyme Assays. The artificial electron donor methyl viologen was used as reductant in all assays reported here. This is because the natural reductants NADPH and NADH (for eukNR from A. niger and T. weissf logii, respectively) interfered with the removal of nitrite from assay subsamples prior to isotope analysis (see supporting text S2.2 of the SI). Any residual nitrite in assay subsamples severely compromises isotope results, as both reactant nitrate and product nitrite are converted to the nitrous oxide (N2O) gas analyte in the denitrifier method used for isotope analysis.4,32 Four initial nitrate concentrations were used in assays of NADPH eukNR from A. niger (100, 250, 500, and 1000 μM). Assays of NADH eukNR from T. weissf logii were conducted at 250 μM nitrate only, for direct comparison with the analogous A. niger assay. The composition of all assays and sampling procedures are provided in supporting text S2.3 of the SI. 2.3. Nitrite Determination and Removal. Nitrite concentrations of assay subsamples were measured immediately upon collection by conversion of nitrite in 100 μL of assay mixture to nitric oxide (NO) gas in hot iodide solution followed by chemiluminescence detection35 on a Teledyne 200E chemiluminescence NOx analyzer. Nitrite was then immediately removed from the remaining subsample by addition of 55 μL 4% (wt/vol) sulfamic acid in 20% (v/v) HCl,36 which yielded the optimum pH of ∼2 for reaction of nitrous acid with sulfamic acid. For short-term sample preservation, the enzyme was denatured by heating samples 5728

dx.doi.org/10.1021/es204593q | Environ. Sci. Technol. 2012, 46, 5727−5735


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Table 1. Nitrate N (15ε) and O (18ε) Isotope Effect Estimates and the Ratio of O and N Isotopic Fractionation (Δδ18O:Δδ15N) for NADPH eukNR from Aspergillus niger and NADH eukNR from Thalassiosira weissf logii initial [NO3−] (μM)

fa

Aspergillus niger 100 assay A 0.30 250 assay A 0.40 500 assay A 0.63 1000 assay A 0.79 weighted mean 100 assay Bc 0.3 250 assay Bc 0.34 500 assay Bc 0.63 weighted mean all A. nigerdatac Thalassiosira weissflogii 250 assay A 0.84 250 assay Bc 0.85 weighted mean all T. weissf logii datad

ε (‰)b

15

26.3 26.9 26.9 26.7 26.6 22.2 24.1 27.4

± ± ± ± ± ± ± ±

R2

0.3 0.3 0.5 2.3 0.2 0.5 0.3 1.2

18

0.9998 0.9998 0.9994 0.9846

23.5 25.0 26.4 28.1 24.9 19.4 22.7 26.4 0.96

0.9982 0.9992 0.9972

27.2 ± 1.8 29.3 ± 2.1

ε (‰)

0.9887 0.9838 1.09 ± 0.03

± ± ± ± ± ± ± ± ±

R2

0.8 0.4 1.1 2.2 0.3 0.6 0.5 1.4 0.01

30.9 ± 2.6 29.9 ± 1.6

Δδ18O:Δδ15N

R2

± ± ± ±

0.9994 0.9997 0.9975 0.9881

0.94 0.95 1.01 1.09

0.01 0.01 0.02 0.04

0.9999 0.9998 0.9994 0.9970

0.9969 0.9981 0.9973

0.91 ± 0.02 0.96 ± 0.01 0.97 ± 0.01

0.9991 0.9997 0.9998

0.9819 0.9915

1.17 ± 0.06 1.06 ± 0.04

0.9936 0.9961

a Fraction of [NO3−] unutilized at final sample point bAll estimates are reported ±1 standard deviation. c[NO3−] for these assays was derived from measured nitrite production; the magnitude of isotope effects derived from these assays is therefore inexact. Only the results of assays where [NO3−]was directly measured are included in the mean reported 15ε and 18ε. dRegressions from all assays are included in the mean Δδ18O:Δδ15N, as the ratio of N and O coupling is independent of [NO3−].

derived by fitting nitrate δ15N and δ18O data to the linear equations:41

to 80 °C for 1 min. Samples were left at room temperature overnight to ensure all nitrite was trapped by sulfamic acid then stored frozen at −20 °C until nitrate isotope analysis.36 Samples were analyzed for nitrate isotopes typically within one day and always within one week. 2.4. Nitrate Isotope Analyses. Nitrate concentrations in assay subsamples were measured by reduction to NO in heated vanadium solution followed by chemiluminescence detection on a Teledyne 200E Chemiluminescence NOx analyzer.37 Reported concentrations are corrected for dilution by the added volume of sulfamic acid. The N and O isotopic composition of nitrate was measured using the “denitrifier” method, where nitrate (and nitrite, when present) is quantitatively converted to N2O gas by denitrifying bacteria that lack the N2O reductase enzyme.4,32 The N and O isotopic composition of the N2O gas is then measured on a gas chromatograph-isotope ratio mass spectrometer. Isotope ratios are reported in delta (δ) notation in units of per mil (‰):

ln(δ15 N + 1) = ln(δ15 N initial + 1) + 15εln(f )

(3)

ln(δ18O + 1) = ln(δ18Oinitial + 1) + 18εln(f )

[NO3−]/[NO3−]initial.

(4) 15

where f = Regression of ln(δ N + 1) or ln(δ18O + 1) on ln( f) yields slopes of 15ε or 18ε. The regressions and their statistical errors were determined by fitting the data with a “least-squares cubic” analysis, a major axis regression analysis where the line is fit by minimizing the sum of the squares of the normal deviates, weighted to account for the different uncertainty associated with the two parameters being compared. The isotope ratio measurements were plotted using a simplified version of the Rayleigh model. δ15N of nitrate was plotted on ln(NO3−) and ln( f) according to the linear equations:

δ15 N sample = (15 N/14 N)sample /(15 N/14 N)reference − 1

(1)

δ15 N = δ15 N initial − 15εln(f )

(5)

δ18Osample = (18O/16O)sample /(18O/16O)reference − 1

(2)

δ15 N = δ15 N initial + 15εln(NO−3 )

(6)

15

14

18

[NO3−]

16

where the N/ N reference is N2 in air and the O/ O reference is Vienna standard mean ocean water (VSMOW). Individual analyses were referenced to injections of N2O from a pure gas cylinder and standardized through comparison to reference material IAEA-N3, with assigned δ15N of 4.7‰38 and δ18O of 25.6‰,39 and reference material USGS-34 (δ15N = −1.8‰ and δ18O = −27.9‰).39 O isotope data were corrected for exchange of oxygen atoms with water during reduction of nitrate to N2O40 by comparing the observed and reported δ18O difference between IAEA-N3 and the reference material USGS34 (assigned δ18O of −27.9‰).39 The standard deviation of replicate measurements of reference materials was less than 0.1‰ for δ15N and 0.3‰ for δ18O (n = 4) within any analysis day. Where samples could be replicated, standard deviation of replicate measurements was less than 0.1‰ for δ15N and 0.3‰ for δ18O (n = 2−4). 2.5. Derivation of N and O Isotope Effects. Estimates of the N and O isotope effects (15ε and 18ε, respectively) were

where is the residual nitrate concentration and the slope of the line approximates 15ε .41 Two assays (“A” and “B”) were conducted at each nitrate concentration (except 1000 μmol L−1) for eukNR from A. niger and T. weissf logii (Table 1). In all “A” assays, the nitrate concentration was directly measured in assay subsamples. In all “B” assays, nitrate was not directly measured but instead calculated based on measured accumulation of nitrite in assay subsamples and the intended initial nitrate concentration (100, 250, or 500 μM). Since calculation of 15ε and 15ε is dependent on nitrate concentration (eqs 3−6 above), the N and O isotope effect estimates associated with “B” assays were therefore subject to greater uncertainty, and they were not used in deriving the weighted mean N and O isotope effects reported for A. niger and T. weissf logii here (Table 1). Data from both “A” and “B” assays were included in determining the ratio of change in δ18O versus δ15N, as this regression is independent of nitrate concentration. 5729



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Figure 1. (a) Nitrate δ15N versus ln[NO3−] for assays of NADPH eukNR from A. niger and NADH eukNR from T. weissf logii. The slope of the linear regression for each assay equals the N isotope effect. (b) Nitrate δ15N versus ln( f), which normalizes the x-axis to fraction of initial nitrate remaining ( f = [NO3−]/[NO3−]initial). In panel b, the δ15N and ln(f) data are normalized to initial values. The slopes and therefore N isotope effects are roughly equivalent for all assays. Table 1 lists the N isotope effect and R2 value associated with each regression. The weighted mean of 15ε estimates from both A. niger and T. weissf logii yields an N isotope effect of 26.6‰ ± 0.2‰ for eukNR.

3. RESULTS Nitrate decreased with time in each assay as it was reduced to nitrite by eukNR (see Figure S1 and supporting text S3.1 of the SI). Nitrate reduction proceeded at a slower rate in the assays of NADH eukNR from T. weissf logii. This is because the enzyme was present at lower concentrations in the T. weissf logii cell homongenates used in these assays, compared to the purified enzyme extracts used in the assays of NADPH eukNR from A. niger. As nitrate decreased, the δ15N and δ18O of the residual nitrate pool increased. The increase in δ15N and δ18O was of similar magnitude. Nitrate δ15N plotted against ln[NO3−] (Figure 1a) and against ln( f) (Figure 1b) conformed to the linear relationship predicted by the Rayleigh model for fractionation in a closed system (eqs 5 and 6, respectively). The slope of the lines approximates the N isotope effect (15ε) (eqs 5 and 6 and Table 1). The slopes and thus N isotope effects were roughly equivalent for all assays regardless of the initial nitrate concentration (Table 1 and Figure 1a,b). The weighted mean for all Assay A results for A. niger NADPH eukNR was 26.6 ± 0.2‰ (±1 standard deviation associated with the mean) (Table 1). The N isotope effect for T. weissf logii NADH eukNR (27.2 ± 1.8‰) was indistinguishable from that measured for NADPH eukNR from A. niger (see supporting text S3.2 of the SI) (Table 1). The weighted mean for all Assay A results for both A. niger and T. weissf logii eukNR was 26.6 ± 0.2‰ (±1 standard deviation associated with the mean), which from this point forward we will report as the N isotope effect for eukNR. The O isotope effect estimate for each assay is also given in Table 1. δ18O and δ15N covaried linearly with a ratio near 1 in all assays (i.e., Δδ18O:Δδ15N ≈ 1) (Figure 2 and Table 1). The mean Δδ18O:Δδ15N for all A. niger assays was 0.96 ± 0.01‰ (weighted mean ±1 standard deviation associated with the mean) (Table 1). The mean for T. weissf logii assays was 1.09 ± 0.03.

Figure 2. The δ18 O change of nitrate plotted against the corresponding δ15N change of nitrate for assays of NADPH eukNR from A. niger and NADH eukNR from T. weissf logii. The δ18O and δ15N data are normalized to initial nitrate δ18O and δ15N values. A line with slope of 1 is shown for reference. The mean slope for all A. niger assays is 0.96 ± 0.01. The mean slope for all T. weissflogii assays is 1.09 ± 0.03. The ratio of O and N isotopic fractionation for individual assays is reported in Table 1.

δ18O covaries linearly with δ15N with a ratio near 1 (i.e., Δδ18O:Δδ15N ≈ 1) during nitrate assimilation.e.g.4,6,7,42 In laboratory cultures of nitrate-assimilating, unicellular marine algae, near equivalent fractionation of N and O isotopes was observed in the intracellular nitrate pool as well as in the ambient medium.6 The δ15N and δ18O of the intracellular pool was elevated relative to the medium.6,22 These linked observations led to the hypothesis that the intracellular enzyme eukNR fractionates N and O isotopes of nitrate with an 18ε:15ε of ∼1.6 We confirm this hypothesis conclusively here (Figure 2 and Table 1). These are the first reliable measurements of both N and O isotope effects for any form of eukNR. A previous study observed a substantially lower O isotope effect and 18ε:15ε for NADH eukNR from corn and Chlorella vulgaris (18ε ≈ 15‰

4. DISCUSSION 4.1. Near Equivalent N and O Isotope Fractionation by eukNR. Both field and laboratory studies have shown that 5730



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and 18ε:15ε ≈ 0.5, Table S1 of the SI).28,29 However, the O isotope measurements in that study were made with an off-line pyrolysis-based method that has since been shown to underestimate nitrate δ18O variations.4,43 Within the monophyletic, closely related family of eukNR enzymes, the most marked division is between the NADPHspecific form of the enzyme unique to fungi and the NADHspecific form found in higher plants and algae.34 We have measured an 18ε:15ε of ∼1 for both the NADPH form (from A. niger) and NADH form (from T. weissf logii) of the enzyme, suggesting this ratio is robust across the eukNR family. The magnitude of N isotope effect also appears to be conserved between forms. The magnitude for the NADH form (27.2 ± 1.8‰) is indistinguishable from that of the NADPH form (26.6‰ ± 0.2‰); the weighted mean of 15ε estimates for eukNR from both A. niger and T. weissf logii yields an N isotope effect of 26.6‰ ± 0.2‰ for eukNR. The coherence in isotopic fractionation between NADH and NADPH eukNR suggests that any differences between these forms of the enzyme do not involve the isotopically sensitive step of nitrate N−O bond rupture or its enzyme-level expression. This finding is consistent with the view that NADH and NADPH eukNR differ substantially only in the domain where NADH or NADPH bind.44 We note that in this study, we used the artificial reductant methyl viologen (MeVi) rather than the natural reductant NAD[P]H. The coherence between our measured values and previous estimates of the NADH-based isotope effect for eukNR (see Table S1 of the SI) suggests that it is appropriate to consider our values representative of what would be observed for NADH-based catalysis in the environment. We address the relative magnitudes of the MeVi-based and NADHbased isotope effects in more detail later in the Discussion. 4.2. Mechanism Underlying Expression of the N and O Isotope Effects for Nitrate Assimilation. The N and O isotope effects for nitrate assimilation, like most biological processes, can vary in magnitude. Because of this, development of a mechanistic (i.e., biochemical and physiological) understanding of the organism-level N and O isotope effects of nitrate assimilation would greatly strengthen environmental applications of nitrate isotope measurements. The net organism-level N isotope effect associated with nitrate assimilation is the result of isotopic fractionation associated with each step in assimilation, εorg, up to the first irreversible step of nitrate reduction. The steps and associated individual isotope effects are uptake (εin), reduction (εNR), and efflux (εout)45 (Figure 3). The organism-level isotope effect (εorg) is approximated by the following:45 εorg = ε in + f (ε NR − εout)

Figure 3. Schematic of isotopic fractionation associated with algal nitrate assimilation. The organism-level N isotope effect associated with nitrate assimilation (i.e., the isotope effect expressed in the environment, εorg) is the result of isotopic fractionation associated with each step in assimilation, up to the first irreversible step of nitrate reduction. The steps and associated individual isotope effects are uptake (εin), enzymatic reduction (εNR), and efflux (εout). Intracellular reduction by nitrate reductase is thought be the dominant fractionating step in nitrate assimilation; if so, efflux of high 15N-nitrate to the medium expresses this fractionation outside the cell.6,14,20,21,23 Observations of (1) elevated δ15N and δ 18O of the internal nitrate pool relative to the ambient medium and (2) equivalent fractionation of N and O isotopes in both internal and ambient nitrate support a reductase-driven model and led to the hypothesis that nitrate reductase fractionates N and O isotopes equally, which is confirmed in this study.

(ii) fractionate N and O isotopes with an enzymatic isotope effect equal to or higher than the maximum organism-level isotope effect observed for nitrate assimilation in culture and field studies. We clearly show equivalent N and O isotopic fractionation by nitrate reductase (Figure 2). Further, the enzymatic isotope effect we observe (26.6 ± 0.2‰) (Figure 1 and Table 1) is significantly higher than any organism-level isotope effects measured to date (16.2‰ is the highest measured for T. weissf logii, 22 23‰ was measured in Phaeodactylum tricornutum21). Thus, our results clearly support a reductase-driven model of organism-level fractionation for nitrate assimilation (see also supporting text S4.1 of the SI). 4.3. Support for Robust Association of Nitrate Assimilation with an 18ε:15ε of ∼1. The association of nitrate assimilation and denitrification with a net organism-level 18 15 ε: ε of ∼1 forms the cornerstone of studies using coupled N and O isotope measurements to separate the effects of cooccurring N cycle processes that have competing N isotope signatures (for example, see refs 3, 8, and 10). Importantly, measuring near equivalent N and O fractionation by eukNR suggests that the association of nitrate assimilation with an 18 15 ε: ε of ∼1 will apply broadly in the environment, for the following reasons. The remaining uncertainty in the biological systematics underlying the observed ratio of ∼1 is the role of nitrate uptake. The ratio of N and O fractionation by uptake is not well constrained. Figure 3 and eq 7 illustrate that as the relative rate of efflux to reduction (i.e., of efflux to uptake) decreases, the magnitude of the organism-level N and O isotope effects decreases, and the influence of uptake on the organism-level N and O isotope effects increases. Near equivalent fractionation of δ18O and δ15N has consistently been measured in association with low N isotope effects (as low as 3.4‰) in field studies and in laboratory studies of eukaryotic organisms.4,6,7,14,42 Now that it is clear that eukNR fractionates N and O isotopes in a ratio near 1, the ratio of ∼1 associated with low organism-level isotope effects can be interpreted in two ways. First, uptake fractionates N and O isotopes in a ratio other than 1 but with a magnitude so low that it has little effect on net fractionation. In

(7)

where f is the relative proportion of efflux to gross uptake ( f = efflux/uptake). Isotopic fractionation associated with nitrate assimilation is thought to originate dominantly from intracellular nitrate reduction.20−23 Efflux of high 15N/14N-nitrate to the ambient medium must then express this fractionation outside the cell, with the degree of environmental expression of the nitrate reductase isotope effect being modulated by f (Figure 3 and eq 7). Previous observations supporting this reductase-driven model of organism-level isotopic fractionation are reported in refs 6, 14, and 20−23. To be consistent with a reductase-driven model of fractionation, we would expect nitrate reductase to (i) fractionate N and O isotopes of nitrate in a ratio of 1 and 5731



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Table 2. Predicted Dependence of N Isotope Effect for eukNR on the Rate of Internal Electron Transfer, Shown by the Relationship between the Observed Isotope Effect for Methyl Viologen-Based Catalysis (εMeVi) and the Observed Isotope Effect for NAD[P]H-Based Catalysis (εNAD[P]H)a

a The use of the artificial reductant methyl viologen simulates a higher rate of internal electron transfer relative to catalysis based on the natural reductant NAD[P]H. bThe * indicates the isotopically sensitive step. 2e− and an arrow indicate at what point electrons are transferred to the Mo center.

this case, the ratio of ∼1 observed in the environment is a direct expression of fractionation by nitrate reductase. Second, nitrate uptake also fractionates N and O isotopes in a ratio of ∼1. In either case, an organism-level 18ε:15ε near 1 will occur at most magnitudes of N isotope effect, and thus an 18ε:15ε near 1 will apply broadly to nitrate assimilation in the environment. 4.4. Insights into the Catalytic Mechanism of eukNR. Isotope effects associated with enzymatic reactions provide constraints on the transition state structure and the relative rates of steps in a reaction.24 Making the standard assumption that the binding and unbinding of substrates to and from enzymes occurs without significant isotope fractionation, the isotope effect observed for an enzymatic reaction is a function of (i) the intrinsic isotope effect associated with the irreversible, isotopically sensitive step and (ii) the extent to which the isotopically sensitive step is rate-limiting, in turn affecting the extent to which prior steps are reversible (the “commitment to catalysis” or “partition factor”).24 The relationship between the enzyme-level observed isotope effect (εobs), the intrinsic isotope effect (εi), and the commitment to catalysis c is given by the following:24

εobs =

εi + c 1+c

εobs =

For a simple reaction involving reversible binding of substrate (S) to enzyme (E) followed by irreversible conversion of substrate to product (P): k1

k2

(10)

The intrinsic isotope effect εi is expressed via dissociation of substrate from the enzyme. As the isotopically sensitive step becomes more rate-limiting (k3 decreases), commitment to catalysis decreases (i.e., more substrate dissociates), and the observed isotope effect increases (eqs 8 and 10). εobs will range from a maximum of εi when c = 0 (k2 ≫ k3) to a minimum of 0 when c → ∞ (k2 ≪ k3) (eqs 8 and 10). For eukNR, reduction of nitrate (specifically, N−O bond rupture) is the isotopically sensitive step.30 Catalysis involves two substrates (NAD[P]H and nitrate) that bind to separate sites or centers on the enzyme. Reductant NAD[P]H binds and donates electrons at an FAD center, nitrate binds and is reduced at a molybdenum (Mo) center, and a heme subunit mediates internal electron transfer between the two.44 Results of kinetic studies suggest that internal electron transfer may be the rate-limiting step to catalysis.46−48 Our measurements of the observed isotope effect for eukNR were made using the artificial reductant methyl viologen. Methyl viologen effectively simulates an increase in the rate of internal electron transfer by donating electrons directly to the Mo center.49 The relationship between the isotope effect for MeVi-based catalysis (εMeVi) and the isotope effect for NAD[P]H-based catalysis (εNAD[P]H) would therefore show the dependence of the net isotope effect on the rate of internal electron transfer. Such a dependence, or lack thereof, can provide insight into the order of steps in catalysis and therefore the nature of the kinetic mechanism for eukNR. We consider three possible reaction sequences at eukNR’s Mo center and the resultant dependence of observed isotope effect on rate of internal electron transfer (Table 2). In case 1, NO3− binds to a reduced Mo center (Table 2). The rate of

(8)

k3

E + S ⇄ ES → EP

εi + k 3/k 2 1 + k 3/ k 2

(9)

c is k3/k2 (see also supporting text S4.2 of the SI), such that eq 8 becomes: 5732



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likely originates with enzymatic nitrate reduction for both processes;5,6 and (iii) eukNR and the dissimilatory NAR enzyme are both molybdoenzymes with similar coordination chemistry at their reaction centers.54 On the basis of these similarities and our results for eukNR, we predict that dissimilatory nitrate reductase NAR also fractionates N and O isotopes in a ratio of 1, although this will clearly require confirmation by direct measurement. The extension of our results to the NAR enzyme is important for understanding the origin of the signature O-to-N ratio of ∼1 associated with marine denitrification.3 It also has significant implications for using nitrate N and O isotopes to study denitrification in freshwater and terrestrial environments. In nitrate isotope data from freshwater systems, δ18O and δ15N typically vary in a ratio well below 1 (i.e., Δδ18O:Δδ15N < 1).13 The observation of Δδ18O:Δδ15N < 1 in freshwater systems has largely been taken as the pure biological signature of dissimilatory nitrate reduction, reviewed in ref 13. This assumption is based on the study discussed earlier that measured an O-to-N ratio of 0.5 for nitrate reductase enzymes28,29,55 using methodology that underestimates changes in δ18O and therefore 18ε:15ε (Table S1 of the SI).4,43 We have shown conclusively here that the O-to-N ratio for eukNR is instead ∼1 and that this likely extends to the dissimilatory NAR enzyme. Our results therefore suggest that the Δδ18O:Δδ15N < 1 observed in freshwater systems is not the pure biological signature of nitrate reduction by NAR and that instead, there are additional processes at work, e.g., overprinting processes such as nitrification (see ref 13 for a review of candidate processes). We note that the periplasmic dissimilatory nitrate reductase enzyme NAP appears to fractionate with an O-to-N ratio of ∼0.6, similar to the ratio observed in freshwater environments, but this auxiliary enzyme is unlikely to catalyze sufficient nitrate reduction in the environment (versus NAR) to propagate this signal.5

internal electron transfer has no effect on the commitment to catalysis, k3/k2, and therefore on the magnitude of the observed isotope effect, such that εMeVi = εNAD[P]H. In case 2, NO3− binds to oxidized Mo (Table 2). Internal electron transfer occurs at the isotopically sensitive step k3, hence its rate has a direct effect on the relative rate of nitrate reduction and dissociation. The increase in the effective rate of internal electron transfer with use of MeVi as reductant leads to an increase in k3 and the commitment to catalysis and therefore a decrease in the observed isotope effect (eq 10), such that εMeVi < εNAD[P]H. Finally, in case 3, NO3− binds to an oxidized Mo center (Table 2). NO3− remains bound while the Mo center is reduced and then is itself reduced (Table 2). As all NO3− bound is reduced, the observed isotope effect is 0. Table S2 of the SI shows how case 1, 2, and 3 relate to the enzyme kinetic mechanisms proposed for eukNR and lists the commitment to catalysis term for each case, which in turn shows the expected dependence of enzymatic isotope effect on [NO3−] and [NAD[P]H]. While we do not yet have data for a direct comparison of εMeVi and εNAD[P]H (see Methods), we can compare our MeVibased isotope effect for T. weissf logii (27.2 ± 1.8‰) with estimates of the NAD[P]H-based eukNR isotope effect from organism-level studies of the same algal species (22‰ and 23.5‰, see Table S1 and supporting text S4.3 of the SI). The data in hand suggest that εMeVi roughly equals (i.e., is not lower than) εNAD[P]H. Direct measurements27−29 or inferred estimates23,50 of εNAD[P]H in other species corroborate this; they generally show an εNAD[P]H equal to or less than our measured values for εMeVi (see Table S1 and supporting text S4.4 of the SI). In the context provided above, this leads to two possible conclusions. The results are consistent with (1) case 1 only, where nitrate binds to reduced Mo, or alternatively, (2) case 1 or case 2 if substrates NO3− and NAD[P]H are in a state of rapid equilibrium with the enzyme, which has been reported in kinetic studies of eukNR.47,51,52 When substrates are in a state of rapid equilibrium, the rate of dissociation of substrates is much greater than the rate of reaction (i.e., k2 ≫ k3). The commitment to catalysis in this case is 0, and the measured isotope effect equals the intrinsic isotope effect associated with N−O bond rupture (εi) at all substrate concentrations (eq 10)53 (see also supporting text S4.5 and S4.6 of the SI). Importantly, if substrates are in rapid equilibrium with the enzyme (see supporting text S4.5 of the SI), then the magnitude of the N isotope effect and ratio of O and N fractionation we measure is that associated with the intrinsic isotope effect for eukNR-mediated NO3− reduction.53 Knowledge of the intrinsic isotope effect and ratio of O and N fractionation for eukNR-mediated N−O bond rupture, in conjunction with molecular modeling, would provide further insight into the nature of the transition state for the eukNR enzyme family. 4.5. Extension to Dissimilatory Nitrate Reductase NAR. Both nitrate assimilation and respiratory nitrate reduction [i.e., denitrification, largely catalyzed by the membrane-bound dissimilatory nitrate reductase (NAR)] impart equivalent fractionation on the N and O isotopes of nitrate.3−6 Here, we studied assimilatory nitrate reductase because, in contrast to the dissimilatory form, sufficient amounts of purified enzyme can be readily procured. There are strong similarities between assimilatory and dissimilatory nitrate reduction: (i) denitrification, like nitrate assimilation, imparts equivalent fractionation on N and O isotopes in laboratory cultures5 and in marine environments, see for example ref 3; (ii) isotopic fractionation

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ASSOCIATED CONTENT

S Supporting Information *

Supporting text S1.1−S4.5, Table S1−S2, and Figure S1. This material is available free of charge via the Internet at http:// pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Phone: +61 3 6232 5222; fax: +61 3 6232 5000; e-mail: [email protected]. Present Addresses ⊥

Department of Marine Sciences, University of Connecticut, 1080 Shennecossett Road, Groton, Connecticut 06340 United States. # Environmental Defense Fund, 257 Park Avenue South, New York, New York 10010 United States. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by a University of Tasmania Postgraduate Scholarship awarded to K.L.K., the Antarctic Climate & Ecosystems Cooperative Research Centre “Ocean Control of CO2” program, and by U.S. NSF CAREER grant OCE-0447570 to D.M.S. Comments from Tom Trull and three anonymous reviewers improved the manuscript. 5733



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Eukaryotic Assimilatory Nitrate Reductase Fractionates N and O

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