D2O Mixtures Research Articles

Probing water-driven nanostructures in an ionic liquid using small- and wide-angle neutron scattering: 1-Dodecyl-3-methylimidazolium iodide

In this study, phase variations within a hydrophilic ionic liquid (IL)–D2O mixture were observed in using small- and wide-angle neutron scattering (SWANS). The specific IL used was 1-dodecyl-3-methylimidazolium iodide ([C12mim][I]). Owing to the large neutron scattering length density of deuterium, D2O nanostructures appeared to be enhanced in the SWANS profile. Moreover, the complicated phase diagram of [C12mim][I]–D2O was determined using SWANS. Evidently, the water-rich region featured nanoconfined water (a water pocket), a smectic A phase, a columnar hexagonal phase as a lyotropic mesophase, a bicontinuous cubic phase as a double gyroid, and a layered crystal phase. Additionally, a double gyroid structure appeared in the narrow water-concentration region.

Water at electrode-electrolyte interfaces: combining HOD vibrational spectra with ab initio-molecular dynamics simulations.

We have undertaken a vibrational study of the structure of interfacial water and its potential dependence using H2O : D2O mixtures to explore the O-H and O-D stretching modes of HOD as well as the bending modes of HOD and H2O. Due to the symmetry reduction, some of the complexity characteristic of the vibrational spectrum of water is removed in HOD. Coupled with potential-dependent ab initio simulations of the gold-water interface, this has enabled a deeper insight into the hydrogen-bond network of interfacial water and into how it is affected by the applied potential. Possibly the most important conclusions of our work are (i) the absence of any ice-like first layer of interfacial water at any potential and (ii) that interfacial water reorients around a stable backbone of hydrogen bonds roughly parallel to the electrode surface. At E > pzc, interfacial water molecules are oriented with the oxygen lone pairs towards the surface and form exclusively or nearly exclusively hydrogen-donating hydrogen bonds with other water molecules. At E < pzc, the oxygen lone pairs instead point away from the surface, but the population of hydrogen-donating water molecules does not vanish. In fact, the population of hydrogen-accepting water molecules only dominates at considerably negative charge densities, due to the weak interaction of the hydrogen atoms of interfacial water molecules with the Au surface.

Sensing the heavy water concentration in an H2O–D2O mixture by solid–solid phononic crystals

A new method based on phononic crystals is presented to detect the concentration of heavy water (D2O) in an H2O–D2O mixture. Results have been obtained and analyzed in the concentration range of 0%–10% and 90%–100% D2O. A proposed structure of tungsten scatterers in an aluminum host is studied. In order to detect the target material, a cavity region is considered as a sound wave resonator in which the target material with different concentrations of D2O is embedded. By changing the concentration of D2O in the H2O–D2O mixture, the resonance frequency undergoes a frequency shift. Each 1% change in D2O concentration in the H2O–D2O mixture causes a frequency change of about 120 Hz. The finite element method is used as the numerical method to calculate and analyze the natural frequencies and transmission spectra of the proposed sensor. The performance evaluation index shows a high Q factor up to 1475758 and a high sensitivity up to 13075, which are acceptable values for sensing purposes. The other figures of merit related to the detection performance also indicate high-quality performance of the designed sensor.

CO2 hydrates phase behaviour and onset nucleation temperatures in mixtures of H2O and D2O: Isotopic effects

In this work, we report the CO2 hydrate phase equilibria in water (H2O), heavy water (D2O), and their binary mixtures following the isochoric pressure search method using a rocking cell apparatus. The phase behaviour was mapped within the temperature and pressure range of 276.32 – 284.80 K and 1.59 – 3.78 MPa, respectively. It was found that there is a difference of ∼ 2 K in the equilibrium line of CO2 hydrates formed in H2O and in D2O, respectively. The hydrate dissociation enthalpies obtained using the Clausius-Clapeyron equation indicate almost similar values formed either in D2O, H2O or their mixtures. These shifts in this equilibrium temperature were compared with the triphasic equilibrium temperature variation estimation obtained using Molecular Dynamics Simulations and a very good agreement with the experimentally obtained values was observed. Further, a constant cooling method was used to obtain the onset temperature of hydrate nucleation for these systems at 3.6 MPa. It has been found that during the cooling ramps, the nucleation always occurred in the vicinity of the temperature of maximum density (TMD) of the systems where water still retains some structuredness. The nucleation experiments also give information about the metastable zone width (MSZW) of the studied systems. The results reported in this work indicate the magnitude of the isotopic effect on CO2 hydrate formation and dissociation that may have implications towards the application of hydrate technology for separation and purification processes.

Combinational Vibration Modes in H2O/HDO/D2O Mixtures Detected Thanks to the Superior Sensitivity of Femtosecond Stimulated Raman Scattering.

Overtones and combinational modes frequently play essential roles in ultrafast vibrational energy relaxation in liquid water. However, these modes are very weak and often overlap with fundamental modes, particularly in isotopologues mixtures. We measured VV and HV Raman spectra of H2O and D2O mixtures with femtosecond stimulated Raman scattering (FSRS) and compared the results with calculated spectra. Specifically, we observed the mode at around 1850 cm-1 and assigned it to H-O-D bend + rocking libration. Second, we found that the H-O-D bend overtone band and the OD stretch + rocking libration combination band contribute to the band located between 2850 and 3050 cm-1. Furthermore, we assigned the broad band located between 4000 and 4200 cm-1 to be composed of combinational modes of high-frequency OH stretching modes with predominantly twisting and rocking librations. These results should help in a proper interpretation of Raman spectra of aqueous systems as well as in the identification of vibrational relaxation pathways in isotopically diluted water.

Analysis of the rectifying separation of H2 O–D2 O mixture into light and heavy water by means of mathematical modeling

Objectives. To apply an analytical method for the calculation of a distillation column for the production of D2O at a two-column Kuhn installation operating under vacuum: to simulate the Kuhn installation in the Hysys software; and to compare experimental and calculated data.Methods. Analytical method for the calculation of distillation columns “from stage to stage,” from the lower theoretical separation stage (TSS) to the upper stage. This method is based on phase equilibrium at the TSS with known data of input flows and component concentrations in the column bottoms. Hysys was used as modeling software.Results. Comparison of the calculation results with Kuhn’s experimental data testified to the high calculation accuracy of the vapor–liquid phase equilibrium for the H2O–D2O mixture at the TSS. The convergence of the D2O material balance for the entire installation was 0.005%. The identification parameter was the number of the column feed plate. Simulation of the Kuhn installation in the Hysys software showed a qualitative agreement of D2O concentrations in material flows. The UNIQUAC (UNIversal QUAsiChemical) model was used to calculate activity coefficients. The found values of the number of theoretical separation stages (NTSS) in both columns, were 88 and 153 taking into account the reboiler and condenser. This is less than the experimental 295 and 400, respectively. The discrepancy can be explained by the increased phase equilibrium H2O constant in the UNIQUAC model. However, the convergence of the material balance in terms of D2O was high and amounted to 1.38·10−6 %. The absolute error of the found concentrations in material flows did not exceed 0.12 mol %.Conclusions. The results obtained indicated the possible use of the Hysys modeling software when searching for and optimizing the operating mode of the block diagram of a cascade of distillation columns with direct and recycle flows to separate a mixture of water into light and heavy water. The final results obtained with regard to the operating mode, inlet and outlet material flows (flow rate, composition, temperature, and pressure drop across the column) are recommended for use in the analytical program for the calculation of the distillation column to refine the NTSS and distribution profile of the concentrations of the H2O and D2O components along the height of the column.

Isotopomeric Elucidation of the Mechanism of Temperature Sensitivity in 59Co NMR Molecular Thermometers.

Understanding the mechanisms governing temperature-dependent magnetic resonance properties is essential for enabling thermometry via magnetic resonance imaging. Herein we harness a new molecular design strategy for thermometry─that of effective mass engineering via deuteration in the first coordination shell─to reveal the mechanistic origin of 59Co chemical shift thermometry. Exposure of [Co(en)3]3+ (1; en = ethylenediamine) and [Co(diNOsar)]3+ (2; diNOsar = dinitrosarcophagine) to mixtures of H2O and D2O produces distributions of [Co(en)3]3+-dn (n = 0-12) and [Co(diNOsar)]3+-dn (n = 0-6) isotopomers all resolvable by 59Co NMR. Variable-temperature 59Co NMR analyses reveal a temperature dependence of the 59Co chemical shift, Δδ/ΔT, on deuteration of the N-donor atoms. For 1, deuteration amplifies Δδ/ΔT by 0.07 ppm/°C. Increasing degrees of deuteration yield an opposing influence on 2, diminishing Δδ/ΔT by -0.07 ppm/°C. Solution-phase Raman spectroscopy in the low-frequency 200-600 cm-1 regime reveals a red shift of Raman-active Co-N6 vibrational modes by deuteration. Analysis of the normal vibrational modes shows that Raman modes produce the largest variation in 59Co δ. Finally, partition function analysis of the Raman-active modes shows that increased populations of Raman modes predict greater Δδ/ΔT, representing new experimental insight into the thermometry mechanism.

Freezing and Thawing of D2O/Sand Mixtures Investigated by Neutron Diffraction

Evolution ice diffraction patterns in mixtures of D2O with quartz sand of three different grain coarseness (100–600, 300–800 and 600–1200 μm) were studied under various temperature regimes by means of neutron diffraction method. The studied structural parameters and characteristics involved the phase composition of specimens, Ih D2O ice lattice parameters, and crystallographic texture of the present phases. Variations in the ice crystallographic texture during the repeated freezing and thawing were observed for all tested sample types, showing an intermittent enhancement of ice and quartz texture indices accompanying the start of specimens cooling. Formation of radial internal stresses is demonstrated by the observed split of (002) and (100) diffraction maxima of ice. Estimated mean internal radial stress values are calculated.

Erratum: Neutron Spin Echo Spectroscopy as a Unique Probe for Lipid Membrane Dynamics and Membrane-Protein Interactions.

Erratum: Neutron Spin Echo Spectroscopy as a Unique Probe for Lipid Membrane Dynamics and Membrane-Protein Interactions.

Ruthenium‐Catalyzed E‐Selective Partial Hydrogenation of Alkynes under Transfer‐Hydrogenation Conditions using Paraformaldehyde as Hydrogen Source

AbstractE‐alkenes were synthesized with up to 100 % E/Z selectivity via ruthenium‐catalyzed partial hydrogenation of different aliphatic and aromatic alkynes under transfer‐hydrogenation conditions. Paraformaldehyde as a safe, cheap and easily available solid hydrogen carrier was used for the first time as hydrogen source in the presence of water for transfer‐hydrogenation of alkynes. Optimization reactions showed the best results for the commercially available binuclear [Ru(p‐cymene)Cl2]2 complex as pre‐catalyst in combination with 2,2‐bis(diphenylphosphino)‐1,1‐binaphthyl (BINAP) as ligand (1 : 1 ratio per Ru monomer to ligand). Mechanistic investigations showed that the origin of E‐selectivity in this reaction is the fast Z to E isomerization of the formed alkenes. Mild reaction conditions plus the use of cheap, easily available and safe materials as well as simple setup and inexpensive catalyst turn this protocol into a feasible and promising stereo complementary procedure to the well‐known Z‐selective Lindlar reduction in late‐stage syntheses. This procedure can also be used for the production of deuterated alkenes simply using d2‐paraformaldehyde and D2O mixtures.

Preferential solvation of carbohydrates in water-trifluoroethanol mixtures: a solvent detected heteronuclear NMR approach.

The present study aims to establish a simple approach involving multi-field multinuclear longitudinal relaxation (R1) analysis of the solvents to decipher solute-solvent interactions during the solvation of model carbohydrates in aqueous trifluoroethanol (TFE) co-solvent systems (TFE:D2O). The behavior of D2O and TFE is monitored around β-CD (β-cyclodextrin) and glucose through R1D (2H) and R1F (19F), respectively. Correlation times (τc) are estimated for D2O and TFE for various % (v/v) compositions of TFE:D2O mixtures. The differential trends of the R1 or τc ratio for D2O and TFE (in the presence and absence of carbohydrates) revealed that both β-CD and glucose undergo selective solvation by TFE in comparison to D2O. Owing to its encapsulation properties, β-CD exhibited a comparatively higher tendency to undergo solvation by TFE than glucose. The maximum transfer of solute bound water to bulk solvent appears in the 20-30% (v/v) TFE range. The current approach emerges as being straightforward in contrast to traditional methods that primarily focus on solute behavior to unravel the preferential solvation dynamics.

Sodium Periodate as a Selective Oxidant for Diclofenac Sodium in Alkaline Medium: A Quantum Chemical Approach

Diclofenac sodium is a well known anti-inflammatory drug. It has also been proclaimed to exhibit adverse effects on aquatic animals through sewage and waste water treatment plants. Kinetic and mechanistic studies of the novel oxidation of diclofenac sodium (DFS) by sodium periodate were discussed with an emphasis on structure and reactivity by using kinetic and computational approach. The proposed work had been studied in alkaline medium at 303 K and at a constant ionic strength of 0.60 mol.dm−3. Formation of [2-(2,6-dicloro-phynylamino)-phenyl]-methanol as the oxidation product of DFS is confirmed with the help of structure elucidation. The active species of catalyst, oxidant and oxidation products were recognized by UV and IR spectral studies. Proton inventory studies in H2O−D2O mixtures had been shown the involvement of a single exchangeable proton of OH− ion in the transition state. All quantum chemical calculations were executed at level of density functional theory (DFT) with B3LYP function using 6-31G (d,p) basis atomic set for the validation of structure, reaction and mechanism. Molecular orbital energies, nonlinear optical properties, bond length, bond angles, reactivity, electrophilic and nucleophilic regions were delineated. Influence of various reactants on rate of chemical reaction were also ascertained and elucidated spectro-photometrically. Activation parameters have been assessed using Arrhenius-Eyring plots. A suitable mechanism consistent with observed kinetic results had been implicated and rate law deduced. Copyright © 2020 by Authors, Published by BCREC Group. This is an open access article under the CC BY-SA License (https://creativecommons.org/licenses/by-sa/4.0).

Gas-Phase Isotopic Fractionation Study of Singly and Doubly Deuterated Isotopologues of Water in the H-D Exchange Reaction by Cavity Ring-Down Spectroscopy.

The underlying mechanisms of the triple-oxygen (16O, 17O, and 18O) isotopic content of deuterated (D) isotopologues of water in H-D exchange reactions in the gas phase remain elusive. Herein, we have demonstrated a high-resolution gas-phase spectral analysis of doubly (D2O) and singly (HDO) deuterated isotopologues of water in the region around 7.8 μm using quantum cascade laser-based cavity ring-down spectroscopy. Isotopic fractionations among doubly and singly deuterated species of water, D216O, HD16O, HD17O, and HD18O, in the gas phase were carried out by probing the fundamental and hot band transitions in the ν2 (bending) mode of D2O and the fundamental ν2 transitions for the other water isotopes. We subsequently investigated the fractionations of different D-enriched water isotopologues for the H-D exchange reaction using various mixtures of D2O in H2O. We explored the potential role of triple-oxygen isotopic contents through enrichments and depletions of HD16O, HD17O, and HD18O, involved in the H-D reaction. Our first clear, direct, and quantitative experimental evidence reveals a new picture of gas-phase isotopic fractionation chemistry in a mixture of light and heavy water (H2O-D2O).

The research of time dependence polymeric membrane swelling in water with various deuterium content

Experiments on the Fourier transform spectrometry of Nafion, a water-swollen polymeric membrane, are described. The transmittance spectra of liquid samples and Nafion, soaked in these samples, were examined, depending on the deuterium content in water within the spectral range 1.8 - 2.15 μm. The experiments were carried out according to two protocols: with the first protocol we studied the dynamics of Nafion swelling in H2O + D2O mixtures for the deuterium concentrations 3 < C < 104 ppm, and with the second protocol we studied the dynamics of swelling in pure heavy water (C = 106 ppm). For liquid mixtures in the concentration range 3 < C < 104 ppm, the transmittance spectra are the same, but for Nafion soaked in these fluids, the corresponding spectra are different. It is shown that, in the range of deuterium contents C = 90 - 500 ppm, the behavior of transmittance of the polymer membrane is non-monotonic. In experiments with the second protocol, the dynamics of diffusion replacement of residual water, which is always present in the bulk of the polymer membrane inside closed cavities (i.e., without access to atmospheric air), was studied.

Half-sandwich Ir(III) and Rh(III) 2,2′-dipyridylamine complexes: Synthesis, characterization and in vitro cytotoxicity against the ovarian carcinoma cells

A series of the half-sandwich Ir(III) and Rh(III) complexes [M(η5-Cpx)(dpa)X]PF6 (M = Ir for 1–6 and Rh for 7–12) containing N-(pyridin-2-yl)pyridin-2-amine (2,2′-dipyridylamine, dpa), pentamethylcyclopentadienyl (Cp*; for 1–5 and 7–11) or 1,2,3,4-tetramethyl-5-phenylcyclopentadienyl (Cpph; for 6 and 12) ring, and various monodentate ligands (X), specifically Cl− (for 1, 6, 7 and 12), Br− (for 2 and 8), I− (for 3 and 9), valproato (VP; for 4 and 10) or 4-phenylbutyrato (PB; for 5 and 11), was prepared. The complexes were thoroughly characterized by elemental analysis, IR and NMR spectroscopy and mass spectrometry. A single-crystal X-ray analysis was performed for complex [Ir(η5-Cpph)(dpa)Cl]PF6 (6), revealing a pseudo-octahedral piano-stool geometry with a bidentate N,N′-coordinated dpa ligand, a penta-hapto coordinated Cpph ring and a monodentate chlorido ligand. The crystal structure of complex 6 is stabilized by N–H⋯F, C–H⋯F, C–H⋯Cl, C–H⋯C and C⋯F non-covalent contacts. Complexes 1–12 were screened for their in vitro cytotoxicity against the A2780 human ovarian carcinoma cell line. The best-performing iridium(III) complex 6 showed markedly higher activity (IC50 = 23.5 μM) than complexes 1–3, 5, 9 and 12, whose IC50 ranged from 68.7 to 87.1 μM. Iridium(III) complex 4 and rhodium(III) complexes 7, 8, 10 and 11 were inactive against the A2780 cells in the tested concentration range (IC50 > 100.0 μM). The chlorido complexes 1, 6, 7 and 12 were studied by 1H NMR spectroscopy for their hydrolytic stability in the 20% DMF-d7/80% D2O and 20% MeOD-d4/80% D2O mixture of solvents, revealing Ir(III) complexes 1 and 6 as stable, while Rh(III) complexes 7 and 12 partially hydrolysed in the used medium. Moreover, hydrophobicity (lipophilicity) of complexes 1–12 was studied by an octanol/water partition (logP).

Imidazolium-Based Grafted Anion Exchange Membranes: Interplay between the Morphology and Anion Transport Behavior

Anion-exchange membranes (AEMs) have been regarded as an alternative to proton-exchange membranes (PEMs) in energy conversion devices, due to the advantage of saving expensive platinum catalysts. However, neither the molecular design nor the property understanding is sufficient for developing AEMs capable of practical fuel cell applications. It is crucial to thoroughly study the current AEMs in terms of microphase structures and conducting properties. Recently, we developed a new graft type of AEMs by radiation-induced grafting method, where imidazolium and styrene monomers were grafted into poly(ethylene-co-tetrafluoroethylene) (ETFE) base films under a dose of 80 kGy from the 60Co γ-ray source (QST, Takasaki), followed by alkylation and ion exchange reactions. These AEMs exhibit well-balanced properties of high ion conductivity (> 100 mS/cm at 80oC) and good stability, which are believed to be controlled by the microphase structure of the membrane. In this report, we investigate the memrbane structure and reveal the relationship between structure and properties, using small-angle neutron scattering (SANS) method. Fig. 1 shows typical SANS intensity profiles, I(q), of pristine ETFE films (profile 1), grafted-ETFE membranes (profile 2), dry AEM (profile 3) and AEM equilibrated in D2O (profile 4) as a function of scattering vector q. The different morphologies which can be deduced from the SANS profiles are also shown in the figure. The experiments confirm the semi-crystalline feature of AEM conserved from the original ETFE base film, which is the key factor for the high membrane mechanical stability, and show the dependence of the structure on hydration. Contrast variation experiments performed on AEM equilibrated in water mixtures of H2O and D2O, further show that graft polymers and water consist the hydrophilic ion channels [1]. Since graft polymers distribute randomly in AEM, well-connected hydrophilic ion channels are feasibly formed, which is the key factor for the high membrane conductivity. Above all, the interplay between the morphology and properties can be characterized as follows: 1) semi-crystalline base film offers good mechanical properties; 2) graft polymers and water form ion channels to promote the conductivity. Figure 1

Dynamics of Nafion membrane swelling in H2O/D2O mixtures as studied using FTIR technique.

Experiments with Fourier transform spectrometry of Nafion, a water-swollen polymeric membrane, are described. The transmittance spectra of liquid samples and Nafion, soaked in these samples, were studied, depending on the deuterium content in water in the spectral range 1.8-2.15 μm. The experiments were carried out using two protocols: in the first protocol we studied the dynamics of Nafion swelling in H2O + D2O mixtures for the deuterium concentrations 3 < C < 104 ppm, and in the second protocol we studied the dynamics of swelling in pure heavy water (C = 106 ppm). For liquid mixtures in the concentration range 3 < C < 104 ppm, the transmittance spectra are the same, but for Nafion soaked in these fluids, the corresponding spectra are different. It is shown that, in the range of deuterium contents C = 90-500 ppm, the behavior of transmittance of the polymer membrane is non-monotonic. In experiments using the second protocol, the dynamics of diffusion replacement of residual water, which is always present in the bulk of the polymer membrane inside closed cavities (i.e., without access to atmospheric air), were studied. The experimentally estimated diffusion coefficient for this process is ≈6·10-11 cm2/s.

Reduced Near-Resonant Vibrational Coupling at the Surfaces of Liquid Water and Ice.

We study the resonant interaction of the OH stretch vibrations of water molecules at the surfaces of liquid water and ice using heterodyne-detected sum-frequency generation (HD-SFG) spectroscopy. By studying different isotopic mixtures of H2O and D2O, we vary the strength of the interaction, and we monitor the resulting effect on the HD-SFG spectrum of the OH stretch vibrations. We observe that the near-resonant coupling effects are weaker at the surface than in the bulk, for both water and ice, indicating that for both phases of water the OH vibrations are less strongly delocalized at the surface than in the bulk.

Anomalous Rate of H+ and D+ Excited-State Proton Transfer (ESPT) in H2O/D2O Mixtures: Irreversible ESPT in 1-Naphthol-4-sulfonate.

We employed steady-state and time-resolved fluorescence techniques to study the rates of excited-state proton and deuteron transfer (ESPT) from an irreversible photoacid, 1-naphthol-4-sulfonate, to solvent mixtures of H2O and D2O. We found that the overall ESPT rate to the solvent mixture does not follow a linear relation with the H2O mole ratio. We used a chemical kinetic model to explain the deviation of the ESPT rate constant from linear behavior with H2O mole ratio. There are three water species in the H2O-D2O mixtures, H2O, D2O, and HOD. There are six rate constants of H+ and D+ transfers to the three species. When the H2O mole ratio before mixing is 0.5, HOD mole ratio in the mixture is 0.5. The ESPT rate to HOD is much smaller than that of H+ transfer to neat H2O and hence the concave shape of the plot of ESPT rate constants versus the H2O molar ratio of the mixtures.

Mechanism of Nitrogenase H2 Formation by Metal-Hydride Protonation Probed by Mediated Electrocatalysis and H/D Isotope Effects.

Nitrogenase catalyzes the reduction of dinitrogen (N2) to two ammonia (NH3) at its active site FeMo-cofactor through a mechanism involving reductive elimination of two [Fe-H-Fe] bridging hydrides to make H2. A competing reaction is the protonation of the hydride [Fe-H-Fe] to make H2. The overall nitrogenase rate-limiting step is associated with ATP-driven electron delivery from Fe protein, precluding isotope effect measurements on substrate reduction steps. Here, we use mediated bioelectrocatalysis to drive electron delivery to the MoFe protein allowing examination of the mechanism of H2 formation by the metal-hydride protonation reaction. The ratio of catalytic current in mixtures of H2O and D2O, the proton inventory, was found to change linearly with the D2O/H2O ratio, revealing that a single H/D is involved in the rate-limiting step of H2 formation. Kinetic models, along with measurements that vary the electron/proton delivery rate and use different substrates, reveal that the rate-limiting step under these conditions is the H2 formation reaction. Altering the chemical environment around the active site FeMo-cofactor in the MoFe protein, either by substituting nearby amino acids or transferring the isolated FeMo-cofactor into a different peptide matrix, changes the net isotope effect, but the proton inventory plot remains linear, consistent with an unchanging rate-limiting step. Density functional theory predicts a transition state for H2 formation where the S-H+ bond breaks and H+ attacks the Fe-hydride, and explains the observed H/D isotope effect. This study not only reveals the nitrogenase mechanism of H2 formation by hydride protonation, but also illustrates a strategy for mechanistic study that can be applied to other oxidoreductase enzymes and to biomimetic complexes.