Nitrogen Isotope Effects Research Articles

Characteristic isotope fractionation patterns in s-triazine degradation have their origin in multiple protonation options in the s-triazine hydrolase TrzN.

s-Triazine herbicides (atrazine, ametryn) are groundwater contaminants which may undergo microbial hydrolysis. Previously, inverse nitrogen isotope effects in atrazine degradation by Arthrobacter aurescens TC1 (i) delivered highly characteristic (13C/12C, 15N/14N) fractionation trends for pathway identification and (ii) suggested that the s-triazine ring nitrogen was protonated in the enzyme s-triazine hydrolase (TrzN) where (iii) TrzN crystal structure and mutagenesis indicated H+-transfer from the residue E241. This study tested the general validity of these conclusions for atrazine and ametryn with purified TrzN and a TrzN-E241Q site-directed mutant. TrzN-E241Q lacked activity with ametryn; otherwise, degradation consistently showed normal carbon isotope effects (εcarbon=-5.0‰±0.2‰ (atrazine/TrzN), εcarbon=-4.2‰±0.5‰ (atrazine/TrzN-E241Q), εcarbon=-2.4‰±0.3‰ (ametryn/TrzN)) and inverse nitrogen isotope effects (εnitrogen=2.5‰±0.1‰ (atrazine/TrzN), εnitrogen=2.1‰±0.3‰ (atrazine/TrzN-E241Q), εnitrogen=3.6‰±0.4‰ (ametryn/TrzN)). Surprisingly, TrzN-E241Q therefore still activated substrates through protonation implicating another proton donor besides E241. Sulfur isotope effects were larger in enzymatic (εsulfur=-14.7‰±1.0‰, ametryn/TrzN) than in acidic ametryn hydrolysis (εsulfur=-0.2‰±0.0‰, pH 1.75), indicating rate-determining C-S bond cleavage in TrzN. Our results highlight a robust inverse 15N/14N fractionation pattern for identifying microbial s-triazine hydrolysis in the environment caused by multiple protonation options in TrzN.

Theoretical predictions of isotope effects versus their experimental values for an example of uncatalyzed hydrolysis of atrazine.

Kinetic isotope effects are one of the most powerful experimental techniques for establishing the nature of a chemical process. However their interpretation very often seeks support from electronic structure calculations in order to get detailed information regarding the transition state which is not experimentally available. For an example of atrazine hydrolysis we have shown how the match between experimentally and theoretically determined magnitudes of carbon, nitrogen and chlorine kinetic isotope effects can be used to discuss the mechanism under different reaction conditions. Two different density functionals combined with the explicit presence of solvent molecules and a continuum solvation model revealed that although the reaction proceeds via the same concerted mechanism regardless of the reaction conditions the transition state structure for an acid and base-catalyzed pathway is different.

Nitrogen isotope effects induced by anammox bacteria.

Nitrogen (N) isotope ratios ((15)N/(14)N) provide integrative constraints on the N inventory of the modern ocean. Anaerobic ammonium oxidation (anammox), which converts ammonium and nitrite to dinitrogen gas (N2) and nitrate, is an important fixed N sink in marine ecosystems. We studied the so far unknown N isotope effects of anammox in batch culture experiments. Anammox preferentially removes (14)N from the ammonium pool with an isotope effect of +23.5‰ to +29.1‰, depending on factors controlling reversibility. The N isotope effects during the conversion of nitrite to N2 and nitrate are (i) inverse kinetic N isotope fractionation associated with the oxidation of nitrite to nitrate (-31.1 ± 3.9‰), (ii) normal kinetic N isotope fractionation during the reduction of nitrite to N2 (+16.0 ± 4.5‰), and (iii) an equilibrium N isotope effect between nitrate and nitrite (-60.5 ± 1.0‰), induced when anammox is exposed to environmental stress, leading to the superposition of N isotope exchange effects upon kinetic N isotope fractionation. Our findings indicate that anammox may be responsible for the unresolved large N isotope offsets between nitrate and nitrite in oceanic oxygen minimum zones. Irrespective of the extent of N isotope exchange between nitrate and nitrite, N removed from the combined nitrite and nitrate (NOx) pool is depleted in (15)N relative to NOx. This net N isotope effect by anammox is superimposed on the N isotope fractionation by the co-occurring reduction of nitrate to nitrite in suboxic waters, possibly enhancing the overall N isotope effect for N loss from oxygen minimum zones.

Community N and O isotope fractionation by sulfide-dependent denitrification and anammox in a stratified lacustrine water column

We investigated the community nitrogen (N) and oxygen (O) isotope effects of fixed N loss in the northern basin of Lake Lugano, where sulfide-dependent denitrification and anammox are the main drivers of suboxic N2 production. A decrease in nitrate (NO3−) concentration toward the redox transition zone (RTZ) at mid-water depth was paralleled by an increase in δ15N and δ18O from approximately 5‰ to >20‰ and from 0‰ to >10‰, respectively. Ammonium (NH4+) concentrations were highest in the near-bottom water and decreased toward the RTZ concomitant with an increase in δ15N–NH4+ from ∼7‰ to >15‰. A diffusion-reaction model yielded N and O isotope enrichment factors that are significantly smaller than isotope effects reported previously for microbial NO3− reduction and NH4+ oxidation (15εNO3≈10‰, 18εNO3≈7‰, and 15εNH4≈10−12‰). For the Lake Lugano north basin, we constrain the apparent under-expression of the N isotope effects to: (1) environmental conditions (e.g., substrate limitation, low cell specific N transformation rates), or (2) low process-specific (chemolithotrophic denitrification and anammox) isotope fractionation. Our results have confirmed the robust nature of the co-linearity between N and O isotope enrichment during microbial denitrification beyond its organotrophic mode. However, the ratio of 18O to 15N enrichment (18εNO3:15εNO3) associated with NO3− reduction in the RTZ was ∼0.89, which is lower than observed in marine environments and in most culture experiments. We propose that chemolithotrophic NO3− reduction in the Lake Lugano north basin was partly catalyzed by the periplasmic dissimilatory nitrate reductase (Nap) (rather than the membrane-bound dissimilatory Nar), which is known to express comparably low 18εNO3:15εNO3 ratios in the ambient NO3− pool. However, NO2− re-oxidation, e.g., during anammox or microaerobic nitrification, could have contributed to the lowered 18O to 15N enrichment ratios. Although we do not yet understand the exact controls on the observed N (and O) isotope fractionation in the Lake Lugano north basin, our study implies that caution is advised when assuming canonical (i.e., high) N isotope effects for NO3− reduction and NH4+ oxidation in natural environments. In Lake Lugano, the community N (and O) isotope effects by sulfide-dependent denitrification and anammox in a natural ecosystem appear to be significantly lower than for organotrophic denitrification and aerobic ammonium oxidation.

A new mechanistic model of δ18O-N2O formation by denitrification

Anaerobic incubations of flooded and non-flooded agricultural soils and stream sediment were conducted with 18O-labelled water to investigate the stable isotope ratios (δ15N and δ18O) of nitrous oxide (N2O) produced from denitrification. The rates of N2O production and δ15N- and δ18O-N2O values were measured. The amount of oxygen exchange (O-exchange) with water, and the nitrogen and oxygen isotope effects (ε) were calculated. The net 15N isotope effect (NO3−→N2O) for denitrification in this study varied from −30‰ to −9‰. The net 18O isotope effect ranged between +32‰ and +60‰ and was negatively correlated to the total fraction of O-exchange, which varied between 0.40 and 0.94.This manuscript describes a new, comprehensive set of mathematical expressions that can be used to model δ18O values of N2O formed by denitrification and calculate the magnitude of O-exchange and the net 18O isotope effect for denitrification. This mathematical approach is compared to another method of approximating O-exchange (Snider et al., 2009), and we show that this older method provides a minimum estimate of O-exchange. Using this mechanistic model, we discuss how N2O consumption, open/closed systems, and variations in the N2O:N2 ratio can influence the observed δ18O-N2O.The net 18O isotope effect for denitrification in this study was partially controlled by the fractions of O-exchange and N2O reduction, which were likely influenced by the actively denitrifying microbial community and the soil moisture. We conclude that δ18O-N2O values are, in many cases, useful tracers of N2O production because they are often higher than the majority of nitrification-derived δ18O-N2O values. The usefulness of δ18O values to apportion sources must be determined on a case-by-case basis.

Isotope Signatures of N2O in a Mixed Microbial Population System: Constraints on N2O Producing Pathways in Wastewater Treatment

We present measurements of site preference (SP) and bulk (15)N/(14)N ratios (δ(15)N(bulk)(N2O)) of nitrous oxide (N(2)O) by quantum cascade laser absorption spectroscopy (QCLAS) as a powerful tool to investigate N(2)O production pathways in biological wastewater treatment. QCLAS enables high-precision N(2)O isotopomer analysis in real time. This allowed us to trace short-term fluctuations in SP and δ(15)N(bulk)(N2O) and, hence, microbial transformation pathways during individual batch experiments with activated sludge from a pilot-scale facility treating municipal wastewater. On the basis of previous work with microbial pure cultures, we demonstrate that N(2)O emitted during ammonia (NH(4)(+)) oxidation with a SP of -5.8 to 5.6 ‰ derives mostly from nitrite (NO(2)(-)) reduction (e.g., nitrifier denitrification), with a minor contribution from hydroxylamine (NH(2)OH) oxidation at the beginning of the experiments. SP of N(2)O produced under anoxic conditions was always positive (1.2 to 26.1 ‰), and SP values at the high end of this spectrum (24.9 to 26.1 ‰) are indicative of N(2)O reductase activity. The measured δ(15)N(bulk)(N2O) at the initiation of the NH(4)(+) oxidation experiments ranged between -42.3 and -57.6 ‰ (corresponding to a nitrogen isotope effect Δδ(15)N = δ(15)N(substrate) - δ(15)N(bulk)(N2O) of 43.5 to 58.8 ‰), which is considerably higher than under denitrifying conditions (δ(15)N(bulk)(N2O) 2.4 to -17 ‰; Δδ(15)N = 0.1 to 19.5 ‰). During the course of all NH(4)(+) oxidation and nitrate (NO(3)(-)) reduction experiments, δ(15)N(bulk)(N2O) increased significantly, indicating net (15)N enrichment in the dissolved inorganic nitrogen substrates (NH(4)(+), NO(3)(-)) and transfer into the N(2)O pool. The decrease in δ(15)N(bulk)(N2O) during NO(2)(-) and NH(2)OH oxidation experiments is best explained by inverse fractionation during the oxidation of NO(2)(-) to NO(3)(-).

Carbon and Nitrogen Isotope Effects Associated with the Dioxygenation of Aniline and Diphenylamine

Dioxygenation of aromatic rings is frequently the initial step of biodegradation of organic subsurface pollutants. This process can be tracked by compound-specific isotope analysis to assess the extent of contaminant transformation, but the corresponding isotope effects, especially for dioxygenation of N-substituted, aromatic contaminants, are not well understood. We investigated the C and N isotope fractionation associated with the biodegradation of aniline and diphenylamine using pure cultures of Burkholderia sp. strain JS667, which can biodegrade both compounds, each by a distinct dioxygenase enzyme. For diphenylamine, the C and N isotope enrichment was normal with ε(C)- and ε(N)-values of -0.6 ± 0.1‰ and -1.0 ± 0.1‰, respectively. In contrast, N isotopes of aniline were subject to substantial inverse fractionation (ε(N) of +13 ± 0.5‰), whereas the ε(C)-value was identical to that of diphenylamine. A comparison of the apparent kinetic isotope effects for aniline and diphenylamine dioxygenation with those from abiotic oxidation by manganese oxide (MnO(2)) suggest that the oxidation of a diarylamine system leads to distinct C-N bonding changes compared to aniline regardless of reaction mechanism and oxidant involved. Combined evaluation of the C and N isotope signatures of the contaminants reveals characteristic Δδ(15)N/Δδ(13)C-trends for the identification of diphenylamine and aniline oxidation in contaminated subsurfaces and for the distinction of aniline oxidation from its formation by microbial and/or abiotic reduction of nitrobenzene.

The nitrogen isotope effect of benthic remineralization-nitrification-denitrification coupling in an estuarine environment

Abstract. The nitrogen (N) stable isotopic composition of pore water nitrate and total dissolved N (TDN) was measured in sediments of the St. Lawrence Estuary and the Gulf of St. Lawrence. The study area is characterized by gradients in organic matter reactivity, bottom water oxygen concentrations, as well as benthic respiration rates. N isotope effects on the water column associated with the benthic exchange of nitrate (εapp) and TDN (εsed) during benthic nitrification-denitrification coupling were investigated. The sediments were a major sink for nitrate and a source of reduced dissolved N (RDN = DON + NH4+). We observed that both the pore water nitrate and RDN pools were enriched in 15N relative to the water column, with increasing δ15N downcore in the sediments. As in other marine environments, the biological nitrate isotope fractionation of net fixed N loss was barely expressed at the scale of sediment-water exchange, with ϵapp values <3‰. The strongest under-expression (i.e. lowest εapp) of the biological N isotope fractionation was observed at the most oxygenated sites with the least reactive organic matter, indicating that, through their control on the depth of the denitrification zone, bottom water oxygen concentrations and the organic matter reactivity can modulate εapp. For the first time, actual measurements of δ15N of pore water RDN were included in the calculations of εsed. We argue that large fractions of the sea-floor-derived DON are reactive and, hence, involved in the development of the δ15N of dissolved inorganic N (DIN) in the water column. In the St. Lawrence sediments, the combined benthic N transformations yield a flux of 15N-enriched RDN that can significantly elevate εsed above εapp. Calculated εsed values were within the range of 4.6 ± 2‰ and were related to organic matter reactivity and oxygen penetration depth in the sediments. ϵsed reflects the δ15N of the N2 lost from marine sediments and thus best describes the isotopic impact of fixed N loss from sediments on the oceanic fixed N pool. Our mean value for εsed is larger than assumed by earlier work, questioning current ideas with regards to the state of balance of the modern N budget.

Nitrogen and oxygen isotope effects of tissue nitrate associated with nitrate acquisition and utilisation in the moss Hypnum plumaeforme.

Mosses are effective accumulators and indicators of N deposition, but the mechanisms of moss N utilisation remain unclear. This study monitored nitrate concentrations ([NO3-]) in solutions supplied to Hypnum plumaeforme Wils. to characterise NO3- uptake from rain events. Concentrations and isotopic ratios (δ15N and δ18O) of residual NO3- in moss tissues were measured to interpret induced NO3- reduction. Noninduced NO3- reduction was inferred from endogenous [NO3-] and isotopic variations that occurred during 65 days of N deprivation. H. plumaeforme scavenges NO3- effectively from supplied solutions. The uptake rate increased with substrate [NO3-] (0.4-3.9mgNL-1) and generally obeyed saturation (Michaelis-Menten) kinetics. The uptake rate was maximised within 60min after receiving NO3-, irrespective of the initial substrate [NO3-]. Lower tissue [NO3-] and greater isotopic enrichment verified the inducibility of nitrate reductase activity (NRA) by NO3- availability, but short-term darkness did not markedly influence moss NO3- uptake or reduction. Significant reduction and isotopic enrichment were detected in moss NO3- reserves during N deprivation, showing 15ε of 12.1‰ and 18ε of 14.4‰. The Δδ15N:Δδ18O ratios of ~1: 1 implied that NRA is the single process driving 15N and 18O fractionations. These results provide new isotopic insights into the nitrate reductase dynamics of the moss.

A Kinetic and Isotope Effect Investigation of the Urease-Catalyzed Hydrolysis of Hydroxyurea

The urease-catalyzed hydrolysis of hydroxyurea is known to exhibit biphasic kinetics, showing a rapid burst phase followed by a slow plateau phase. Kinetic isotope effects for both phases of this reaction were measured at pH 6.0 and 25 °C. The observed nitrogen isotope effects for the ammonia leaving group [(15)(V/K)(NH(3))] were 1.0016 ± 0.0005 during the burst phase and 1.0019 ± 0.0007 during the plateau phase, while those for the hydroxylamine leaving group [(15)(V/K)(NH(2)OH)] were 1.0013 ± 0.0005 for the burst phase and 1.0022 ± 0.0003 for the plateau phase. These isotope effects are consistent with a rate-determining step that occurs prior to breaking either of the two possible C-N bonds. The observed carbonyl carbon isotope effects [(13)(V/K)] were 1.0135 ± 0.0003 during the burst phase and 1.0178 ± 0.0003 during the plateau phase. The similarity of the magnitude of the carbon isotope effects argues for formation of a common intermediate during both phases.

Deuterium isotope effects on 13C and 15N chemical shifts of intramolecularly hydrogen-bonded enaminocarbonyl derivatives of Meldrum’s and Tetronic acid

Secondary deuterium isotope effects on 13C and 15N nuclear shieldings in a series of cyclic enamino-diesters and enamino-esters and acyclic enaminones and enamino-esters have been examined and analysed using NMR and DFT (B3LYP/6-31G(d,p)) methods. One-dimensional and two-dimensional NMR spectra of enaminocarbonyl and their deuterated analogues were recorded in CDCl 3 and CD 2Cl 2 at variable temperatures and assigned. 1 J NH coupling constants for the derivatives of Meldrum’s and tetronic acids reveal that they exist at the NH-form. It was demonstrated that deuterium isotope effects, for the hydrogen bonded compounds, due to the deuterium substitution at the nitrogen nucleus lead to large one-bond isotope effects at nitrogen, 1Δ 15N(D), and two-bond isotope effects on carbon nuclei, 2ΔC(ND), respectively. A linear correlations exist between 2ΔC(ND) and 1Δ 15N(D) whereas the correlation with δNH is divided into two. A good agreement between the experimentally observed 2ΔC(ND) and calculated dσ 13C/dR NH was obtained. A very good correlation between calculated NH bond lengths and observed NH chemical shifts is found. The observed isotope effects are shown to depend strongly on Resonance Assisted Hydrogen bonding.

Modeling of Isotope Effects on Binding Oxamate to Lactic Dehydrogenase

A new crystal structure of the rabbit muscle L-lactic dehydrogenase (PDB code 3H3F) has been determined. The independent unit of this structure contains two tetramers, each of them with a unique constitution of two active sites with the open loop conformation and two with the loops closed over the actives sites. On the basis of this structure, interactions of an inhibitor, oxamate anion, with the protein have been modeled using different hybrid schemes that involved B3LYP/6-31++G(d,p) DFT theory level in the QM layer. In ONIOM calculations, either Amber (QM:MM) or one of the three semiempirical parametrizations, AM1, PM3, and RM1 (QM:QM) was used, while in the traditional QM/MM scheme, the OPLS-AA force field was used for the outer layer. Normal modes of vibrations of oxamate in aqueous solution and in the active site of the enzyme were used to calculate binding isotope effects. On the basis of the comparison of the values obtained theoretically with those experimentally determined for the oxygen atoms of the carboxylic group of oxamate it was concluded that the DFT/OPLS-AA scheme, applied to the dimer consisting of two chains, one with the open loop and the other with the closed loop conformation, provides the best description of the active site. Calculations of the binding isotope effects of the other atoms of oxamate suggest that nitrogen isotope effect may be useful for the experimental differentiation between open and closed loop conformations.

Isotopologue signatures of N2O produced by denitrification in soils

We investigated oxygen and site‐specific nitrogen isotope effects of N2O produced in the NO3−‐to‐N2O step of denitrification. Arable sand and silt loam soils with varying NO3− availability were incubated under N2/C2H2 atmosphere in order to establish anaerobic conditions and to block N2O reduction. Dual isotope and isotopomer ratios of emitted N2O were determined by analysis of δ18O, average δ15N (δ15Nbulk) and 15N site preference (SP is equal to difference in δ15N between the central and peripheral N positions of the asymmetric N2O molecule). The average N enrichment factor of the NO3−‐to‐N2O step ranged from −47.9 to −53.6‰, which is between the reported ranges of enrichment factors of nitrification and denitrification. SP varied with time, and mean values were between 3.1 and 8.9‰, which is higher compared to SP reported from pure cultures of denitrifiers but lower compared to nitrifiers and fungal denitrifiers. This shows that SP of N2O produced in soils might be seen as a semiquantitative indicator for the different pathways of N2O production but not specific enough to quantify the relative contribution of denitrification to the total N2O flux in systems where several source processes are important. δ18O of N2O from all treatments was less variable compared to δ18O of soil NO3−, indicating that there was a relatively large O exchange with water during N2O formation.

Inverse kinetic isotope fractionation during bacterial nitrite oxidation

Natural abundance stable isotopes in nitrate ( NO 3 - ), nitrite ( NO 2 - ), and nitrous oxide (N 2O) have been used to better understand the cycling of nitrogen in marine and terrestrial environments. However, in order to extract the greatest information from the distributions of these isotopic species, the kinetic isotope effects for each of the relevant microbial reactions are needed. To date, kinetic isotope effects for nitrite oxidation and anaerobic ammonium oxidation (anammox) have not been reported. In this study, the nitrogen isotope effect was measured for microbial nitrite oxidation to nitrate. Nitrite oxidation is the second step in the nitrification process, and it plays a key role in the regeneration of nitrate in the ocean. Surprisingly, nitrite oxidation occurred with an inverse kinetic isotope effect, such that the residual nitrite became progressively depleted in 15N as the reaction proceeded. Three potential explanations for this apparent inverse kinetic isotope effect were explored: (1) isotope exchange equilibrium between nitrite and nitrous acid prior to reaction, (2) reaction reversibility at the enzyme level, and (3) true inverse kinetic fractionation. Comparison of experimental data to ab initio calculations and theoretical predictions leads to the conclusion that the fractionation is most likely inverse at the enzyme level. Inverse kinetic isotope effects are rare, but the experimental observations reported here agree with kinetic isotope theory for this simple N–O bond-forming reaction. Nitrite oxidation is therefore fundamentally different from all other microbial processes in which N isotope fractionation has been studied. The unique kinetic isotope effect for nitrite oxidation should help to better identify its role in the cycling of nitrite in ocean suboxic zones, and other environments in which nitrite accumulates.

Insights into the Mechanism of Flavoprotein-Catalyzed Amine Oxidation from Nitrogen Isotope Effects on the Reaction of N-Methyltryptophan Oxidase

The mechanism of N-methyltryptophan oxidase, a flavin-dependent amine oxidase from Escherichia coli, was studied using a combination of kinetic isotope effects and theoretical calculations. The 15(kcat/Km) kinetic isotope effect for sarcosine oxidation is pH-dependent with a limiting value of 0.994-0.995 at high pH. Density functional theory calculations on model systems were used to interpret these isotope effects. The isotope effects are inconsistent with proposed mechanisms involving covalent amine-flavin adducts but cannot by themselves conclusively distinguish between some discrete electron-transfer mechanisms and a direct hydride-transfer mechanism, although the latter mechanism is more consistent with the energetics of the reaction.

Viewpoint: Isotopic fractionation by plant nitrate reductase, twenty years later.

Plant nitrate reductase, the enzyme that reduces nitrate (NO3-) to nitrite (NO2-), is known to fractionate N isotopes, depleting nitrite in 15N compared with substrate nitrate. Nearly 20 years ago, the nitrogen isotope effect associated with this reaction was found to be around 1.015. However, the relationships between the isotope effect and the mechanism of the reaction have not yet been examined in the light of recent advances regarding the catalytic cycle and enzyme structure. We thus give here the mathematical bases of the 14N / 15N and also the 16O / 18O isotope effects as a function of reaction rates. Enzymatic nitrate reduction involves steps other than NO3- reduction itself, in which the oxidation number of N changes from +V (nitrate) to +III (nitrite). Using some approximations, we give numerical estimates of the intrinsic N and O isotope effects and this leads us to challenge the assumptions of nitrate reduction itself as being a rate-limiting step within the nitrate reductase reaction, and of the formation of a bridging oxygen as a reaction intermediate.

Kinetic isotope effects of nitrogen and hydrogen in reaction of N- tert-butyl- P-phenylphosphonamidothioic acid with alcohols

Nitrogen and hydrogen isotope effects for the reaction of N-tert-butyl- P-phenylphosphonamidothioic acid 1 with alcohols (methanol, butanol, iso-propanol, tert-butanol) were measured in dichloromethane at 30 °C. The observed nitrogen isotope effect k 14/ k 15 is only slightly sensitive to a steric hindrance of the alcohol [1.0070 ± 0.0002 (MeOH), 1.0074 ± 0.0004 (BuOH), 1.0062 ± 0.0004 (Pr i OH), 1.0087 ± 0.0007 (Bu t OH)]. The pre-equilibrium step, with proton transfer from oxygen to nitrogen was proved by the inverse hydrogen effect k ROH/ k ROD[0.778 ± 0.052 (MeOH), 0.863 ± 0.063 (BuOH), 0.883 ± 0.080 (Pr i OH), 0.746 ± 0.084 (Bu t OH)]. The experimental values are consistent with theoretical results of semiempirical calculations on PM3 level for an elimination–addition mechanism and metathiophosphonate PhPSO intermediacy. For the reaction with methanol the addition–elimination mechanism is also possible.

Calculations of substituent and solvent effects on the kinetic isotope effects of Menshutkin reactions.

Nitrogen, deuterium, halogen, and carbon kinetic isotope effects have been modeled for the Menshutkin reaction between methyl halides and substituted N,N-dimethylaniline at the HF/6-31G(d) level of theory augmented by the C-PCM continuum solvent model for several solvents. Systematic changes in geometries of the transition states and Gibbs free energies of activation have been found with phenyl ring substituents, solvent, and the leaving group. Kinetic isotope effects also change systematically; however, these changes are predicted to be small, inside the usual precision of the experimental measurements. On the contrary, no correlation has been found between the kinetic isotope effects and the Hammett constants for para substituents. Thus opposite to previous assumptions, our results indicate that kinetic isotope effects on the Menshutkin reaction cannot be used to predict the position of the transition state on the reaction coordinate.

Modelling nitrogen and oxygen isotope fractionation during denitrification in a lacustrine redox-transition zone

The stable isotope composition (δ 15N and δ 18O) of nitrate was measured during Summer 1999 in the anaerobic hypolimnion of eutrophic Lake Lugano (Switzerland). Denitrification was demonstrated by a progressive nitrate depletion coupled to increasing δ 15N and δ 18O values for residual nitrate. Maximum δ 15N and δ 18O values amounted to 27.2 and 15.7‰, respectively. 15N and 18O enrichment factors for denitrification (ε) were estimated using a closed-system model and a dynamic diffusion-reaction model. Using the Rayleigh equation (closed-system approach), we obtained ε values of −11.2 and −6.6‰ for nitrogen and oxygen, respectively. The average ε values derived using the diffusion-reaction model were determined to be −20.7 ± 3.8 for nitrogen and −11.0 ± 1.7 for oxygen. Both N and O isotope fractionation appeared to be lower when denitrification rates where high, possibly in association with high organic carbon availability. In addition, variations in the isotope effects may be attributed to the variable importance of sedimentary denitrification having only a small isotope effect on the water column. The combined measurement of N and O isotope ratios in nitrate revealed that coupled nitrification-denitrification in the open-water was of minor importance. This is the first study of nitrogen and oxygen isotope effects associated with microbial denitrification in a natural lake. Moreover, this study confirms the high potential of δ 18O of nitrate as a valuable biogeochemical tracer in aquatic systems, complementing nitrate δ 15N.

Superconducting characteristics in electron-doped layered hafnium nitride:15Nisotope effect studies

We report ${}^{15}\mathrm{N}$ nuclear magnetic resonance (NMR) and the nitrogen-isotope effect studies on c-axis oriented sample of Li-doped HfNCl with superconducting transition temperature ${T}_{c}\ensuremath{\sim}25.5\mathrm{K}.$ ${}^{15}\mathrm{N}$ NMR Knight shift decreases toward zero below ${T}_{c},$ giving evidence that the pairing symmetry is an even-parity spin singlet, and that the nitrogen site plays an important role in the occurrence of superconductivity. A nitrogen isotope shift is found to be quite small, $\ensuremath{\Delta}{T}_{c}\ensuremath{\sim}0.1\mathrm{K}$ $({\ensuremath{\alpha}}_{N}=0.07\ifmmode\pm\else\textpm\fi{}0.02),$ which cannot reproduce such a high ${T}_{c}$ within the traditional BCS framework.