Oxygen-18 Kinetic Isotope Effects Research Articles

Oxygen Kinetic Isotope Effects in the Thermal Decomposition and Reduction of Ammonium Diuranate.

Oxygen stable isotopes in uranium oxides processed through the nuclear fuel cycle may have the potential to provide information about a material’s origin and processing history. However, a more thorough understanding of the fractionating processes governing the formation of signatures in real-world samples is still needed. In this study, laboratory synthesis of uranium oxides modeled after industrial nuclear fuel fabrication was performed to follow the isotope fractionation during thermal decomposition and reduction of ammonium diuranate (ADU). Synthesis of ADU occurred using a gaseous NH3 route, followed by thermal decomposition in a dry nitrogen atmosphere at 400, 600, and 800 °C. The kinetic impact of heating ramp rates on isotope effects was explored by ramping to each decomposition temperature at 2, 20, and 200 °C min–1. In addition, ADU was reduced using direct (ramped to 600 °C in a hydrogen atmosphere) and indirect (thermally decomposed to U3O8 at 600 °C, then exposed to a hydrogen atmosphere) routes. The bulk oxygen isotope composition of ADU (δ18O = −16 ± 1‰) was very closely related to precipitation water (δ18O = −15.6‰). The solid products of thermal decomposition using ramp rates of 2 and 20 °C min–1 had statistically indistinguishable oxygen isotope compositions at each decomposition temperature, with increasing δ18O values in the transition from ADU to UO3 at 400 °C (δ18OUO3 – δ18OADU = 12.3‰) and the transition from UO3 to U3O8 at 600 °C (δ18OU3O8 – δ18OUO3 = 2.8‰). An enrichment of 18O attributable to water volatilization was observed in the low temperature (400 °C) product of thermal decomposition using a 200 °C min–1 ramp rate (δ18OUO3 – δ18OADU = 9.2‰). Above 400 °C, no additional fractionation was observed as UO3 decomposed to U3O8 with the rapid heating rate. Indirect reduction of ADU produced UO2 with a δ18O value 19.1‰ greater than the precipitate and 4.0‰ greater than the intermediate U3O8. Direct reduction of ADU at 600 °C in a hydrogen atmosphere resulted in the production of U4O9 with a δ18O value 17.1‰ greater than the precipitate. Except when a 200 °C min–1 ramp rate is employed, the results of both thermal decomposition and reduction show a consistent preferential enrichment of 18O as oxygen is removed from the original precipitate. Hence, the calcination and reduction reactions leading to the production of UO2 will yield unique oxygen isotope fractionations based on process parameters including heating rate and decomposition temperature.

Predicting nitrogen and oxygen kinetic isotope effects of nitrate reduction by periplasmic dissimilatory nitrate reductase

Kinetic isotope effect (KIE) reveals the transition state structure of an elementary reaction and can be predicted by quantum chemical calculation. Density Functional Theory calculation of an enzymatic reaction with large numbers of atoms is computationally prohibitive, especially if explicit solvent effect is considered. Cutoff method, which simplifies an entire molecule to a cluster around a target position, can simplify position-specific equilibrium isotope effect calculation for a large organic molecule. It should also be applicable to KIE calculation of an enzymatic reaction and allow us to introduce explicit solvent molecules to the system. If this treatment is feasible and trustable, it will provide an efficient method to estimate a number of KIEs produced by enzyme reactions. Obviously, its robustness must be tested. Here, using NO3− reduction by the active site of periplasmic dissimilatory nitrate reductase (Nap) in Rhodobacter sphaeroides as an example, we built 17 models to test the influence of cutoff size, implicit, and explicit solvent effects on KIE calculation. The results show that to estimate the KIE value of an enzymatic reaction accurately and efficiently, we can first simplify the reaction model to a cutoff model with 3 proximal bonds to the active position. Then, incorporating implicit-plus-explicit solvent models can simulate a reaction environment more realistically, which is necessary for accuracy. Our calculated lnKIE values for nitrate Nap reduction at 25 °C are −32.4 ± 1.8‰ for 15N and −20.9 ± 0.4 ‰ for 18O, respectively, with a ln18KIE/ln15KIE ratio of 0.65 ± 0.05. Although additional reservoir-transport processes need to be considered, our calculation results are consistent with calibrated isotope effects from laboratory experiments, suggesting that the transition state we calculated depicts the general reaction mechanism of NO3− reduction by Nap.

Ab initio study of nitrogen and position-specific oxygen kinetic isotope effects in the NO + O3 reaction

Ab initio calculations have been carried out to investigate nitrogen (k15/k14) and position-specific oxygen (k17/k16O & k18/k16) kinetic isotope effects (KIEs) for the reaction between NO and O3 using CCSD(T)/6-31G(d) and CCSD(T)/6-311G(d) derived frequencies in the complete Bigeleisen equations. Isotopic enrichment factors are calculated to be -6.7‰, -1.3‰, -44.7‰, -14.1‰, and -0.3‰ at 298 K for the reactions involving the 15N16O, 14N18O, 18O16O16O, 16O18O16O, and 16O16O18O isotopologues relative to the 14N16O and 16O3 isotopologues, respectively (CCSD(T)/6-311G(d)). Using our oxygen position-specific KIEs, a kinetic model was constructed using Kintecus, which estimates the overall isotopic enrichment factors associated with unreacted O3 and the oxygen transferred to NO2 to be -19.6‰ and -22.8‰, respectively, (CCSD(T)/6-311G(d)) which tends to be in agreement with previously reported experimental data. While this result may be fortuitous, this agreement suggests that our model is capturing the most important features of the underlying physics of the KIE associated with this reaction (i.e., shifts in zero-point energies). The calculated KIEs will useful in future NOx isotopic modeling studies aimed at understanding the processes responsible for the observed tropospheric isotopic variations of NOx as well as for tropospheric nitrate.

Oxygen kinetic isotope effects in selenate during microbial reduction

The redox cycling of selenium oxyanions, elemental selenium, and selenides within water resources has implications for Se bioavailability and ecotoxicity. Dual stable isotope analysis of Se and O may provide important information for interpreting environmental Se transformations. Stable Se isotope systematics within the Se redox cycle has been well characterized within the literature, however concomitant oxygen isotope composition requires additional investigation. This study reports the O isotope fractionation of selenate (SeO42−) during microbial reduction by the dissimilatory Se-reducing bacterium Sulfurospirillum barnesii SES-3. Microbial reduction experiments were conducted under various conditions in order to investigate the range of 18O enrichment factors (εO) in selenate. The reduction of selenate to selenite coupled to the oxidation of lactate to acetate resulted in an 18O kinetic isotope effect with εO values 1.5–5.8‰. Greater 18O enrichment was observed with increasing pH, but no correlation was found between εO and reduction rates, lactate availability, or cell density. εO values from biotic reduction by S. barnesii here are significantly less than those observed for abiotic reduction with Fe(II)-rich minerals reported in the literature, and this difference could be explained by a diffusion limitation during enzymatic reduction. Our results expand the isotope systematics of the selenium redox cycle and suggest εO has potential usefulness as indicators for in situ selenate reduction.

Oxygen-18 Kinetic Isotope Effects of Nonheme Iron Enzymes HEPD and MPnS Support Iron(III) Superoxide as the Hydrogen Abstraction Species.

Nonheme iron oxygenases that carry out four-electron oxidations of substrate have been proposed to employ iron(III) superoxide species to initiate this reaction [Paria, S.; Que, L.; Paine, T. K. Angew. Chem. Int. Ed.2011, 50, 11129]. Here we report experimental evidence in support of this proposal. 18O KIEs were measured for two recently discovered mononuclear nonheme iron oxygenases: hydroxyethylphosphonate dioxygenase (HEPD) and methylphosphonate synthase (MPnS). Competitive 18O KIEs measured with deuterated substrates are larger than those measured with unlabeled substrates, which indicates that C–H cleavage must occur before an irreversible reductive step at molecular oxygen. A similar observation was previously used to implicate copper(II) superoxide in the H-abstraction reactions catalyzed by dopamine β-monooxygenase [Tian, G. C.; Klinman, J. P. J. Am. Chem. Soc.1993, 115, 8891] and peptidylglycine α-hydroxylating monooxygenase [Francisco, W. A.; Blackburn, N. J.; Klinman, J. P. Biochemistry2003, 42, 1813].

Online oxygen kinetic isotope effects using membrane inlet mass spectrometry can differentiate between oxidases for mechanistic studies and calculation of their contributions to oxygen consumption in whole tissues.

The reduction chemistry of molecular oxygen underpins the energy metabolism of multicellular organisms, liberating free energy needed to catalyze a plethora of enzymatic reactions. Measuring the isotope signatures of (16)O and (18)O during O2 reduction can provide insights into both kinetic and equilibrium isotope effects. However, current methods to measure O2 isotope signatures are time-consuming and disruptive. This paper describes the application of membrane inlet mass spectrometry to determine the oxygen isotope discrimination of a range of O2-consuming reactions, providing a rapid and convenient method for determining these values. A survey of oxygenase and oxidase reactions provides new insights into previously uncharacterized amino acid oxidase enzymes. Liquid and gas phase measurements show the ease of assays using this approach for purified enzymes, biological extracts and intact tissues.

Competitive oxygen-18 kinetic isotope effects expose O–O bond formation in water oxidation catalysis by monomeric and dimeric ruthenium complexes

Competitive 18O KIEs on water oxidation catalysis provide a probe of transition states for O–O bond formation.

Experimental and Computational Evidence of Metal-O2 Activation and Rate-Limiting Proton-Coupled Electron Transfer in a Copper Amine Oxidase

The mechanism of O(2) reduction by copper amine oxidase from Arthrobacter globiformus (AGAO) is analyzed in relation to the cobalt-substituted protein. The enzyme utilizes a tyrosine-derived topaquinone cofactor to oxidize primary amines and reduce O(2) to H(2)O(2). Steady-state kinetics indicate that amine-reduced CuAGAO is reoxidized by O(2) >10(3) times faster than the CoAGAO analogue. Complementary spectroscopic studies reveal that the difference in the second order rate constant, k(cat)/K(M)(O(2)), arises from the more negative redox potential of Co(III/II) in relation to Cu(II/I). Indistinguishable competitive oxygen-18 kinetic isotope effects are observed for the two enzymes and modeled computationally using a calibrated density functional theory method. The results are consistent with a mechanism where an end-on (η(1))-metal bound superoxide is reduced to an η(1)-hydroperoxide in the rate-limiting step.

Studies of the Di-iron(VI) Intermediate in Ferrate-Dependent Oxygen Evolution from Water

Molecular oxygen is produced from water via the following reaction of potassium ferrate (K(2)FeO(4)) in acidic solution: 4[H(3)Fe(VI)O(4)](+) + 8H(3)O(+) → 4Fe(3+) + 3O(2) + 18H(2)O. This study focuses upon the mechanism by which the O-O bond is formed. Stopped-flow kinetics at variable acidities in H(2)O and D(2)O are used to complement the analysis of competitive oxygen-18 kinetic isotope effects ((18)O KIEs) upon consumption of natural abundance water. The derived (18)O KIEs provide insights concerning the identity of the transition state. Water attack (WA) and oxo-coupling (OC) transition states were evaluated for various reactions of monomeric and dimeric ferrates using a calibrated density functional theory protocol. Vibrational frequencies from optimized isotopic structures are used here to predict (18)O KIEs for comparison to experimental values determined using an established competitive isotope-fractionation method. The high level of agreement between experimental and theoretic isotope effects points to an intramolecular OC mechanism within a di-iron(VI) intermediate, consistent with the analysis of the reaction kinetics. Alternative mechanisms are excluded based on insurmountably high free energy barriers and disagreement with calculated (18)O KIEs.

Oxygen Kinetic Isotope Effects upon Catalytic Water Oxidation by a Monomeric Ruthenium Complex

Oxygen isotope fractionation is applied for the first time to probe the catalytic oxidation of water using a widely studied ruthenium complex, [Ru(II)(tpy)(bpy)(H(2)O)](ClO(4))(2) (bpy = 2,2'-bipyridine; tpy = 2,2';6",2"-terpyridine). Competitive oxygen-18 kinetic isotope effects ((18)O KIEs) derived from the ratio of (16,16)O(2) to (16,18)O(2) formed from natural-abundance water vary from 1.0132 ± 0.0005 to 1.0312 ± 0.0004. Experiments were conducted with cerium(IV) salts at low pH and a photogenerated ruthenium(III) tris(bipyridine) complex at neutral pH as the oxidants. The results are interpreted within the context of catalytic mechanisms using an adiabatic formalism to ensure the highest barriers for electron-transfer and proton-coupled electron-transfer steps. In view of these contributions, O-O bond formation is predicted to be irreversible and turnover-limiting. The reaction with the largest (18)O KIE exhibits the greatest degree of O-O coupling in the transition state. Smaller (18)O KIEs are observed due to multiple rate-limiting steps or transition-state structures which do not involve significant O-O motion. These findings provide benchmarks for systematizing mechanisms of O-O bond formation, the critical step in water oxidation by natural and synthetic catalysts. In addition, the measurements introduce a new tool for calibrating computational studies using relevant experimental data.

Experimental and Computational Investigations of Oxygen Reactivity in a Heme and Tyrosyl Radical-Containing Fatty Acid α-(Di)oxygenase

Rice α-(di)oxygenase mediates the regio- and stereospecific oxidation of fatty acids using a persistent catalytic tyrosyl radical. Experiments conducted in the physiological O(2) concentration range, where initial hydrogen atom abstraction from the fatty acid occurs in a kinetically reversible manner, are described. Our findings indicate that O(2)-trapping of an α-carbon radical is likely to reversibly precede reduction of a 2-(R)-peroxyl radical intermediate in the first irreversible step. A mechanism of concerted proton-coupled electron transfer is proposed on the basis of natural abundance oxygen-18 kinetic isotope effects, deuterium kinetic isotope effects, and calculations at the density functional level of theory, which predict a polarized transition state in which electron transfer is advanced to a greater extent than proton transfer. The approach outlined should be useful for identifying mechanisms of concerted proton-coupled electron transfer in a variety of oxygen-utilizing enzymes.

Oxygen Isotope Effects as Probes of Electron Transfer Mechanisms and Structures of Activated O2

Competitively determined oxygen ((18)O) isotope effects can be powerful probes of chemical and biological transformations involving molecular oxygen as well as superoxide and hydrogen peroxide. They play a complementary role to crystallography and spectroscopy in the study of activated oxygen intermediates by forging a link between electronic/vibrational structure and the bonding that occurs within ground and transition states along the reaction coordinate. Such analyses can be used to assess the plausibility of intermediates and their catalytic relevance in oxidative processes. This Account describes efforts to advance oxygen kinetic isotope effects ((18)O KIEs) and equilibrium isotope effects ((18)O EIEs) as mechanistic probes of reactive, oxygen-derived species. We focus primarily on transition metal mediated oxidations, outlining both advances over the past five years and current limitations of this approach. Computational methods are now being developed to probe transition states and the accompanying kinetic isotope effects. In particular, we describe the importance of using a full-frequency model to accurately predict the magnitudes as well as the temperature dependence of the isotope effects. Earlier studies have used a "cut-off model," which employs only a few isotopic vibrational modes, and such models tend to overestimate (18)O EIEs. Researchers in mechanistic biological inorganic chemistry would like to differentiate "inner-sphere" from "outer-sphere" reactivity of O(2), a designation that describes the extent of the bonding interaction between metal and oxygen in the transition state. Though this problem remains unsolved, we expect that this isotopic approach will help differentiate these processes. For example, comparisons of (18)O KIEs to (18)O EIEs provide benchmarks that allow us to calibrate computationally derived reaction coordinates. Once the physical origins of heavy atom isotope effects are better understood, researchers will be able to apply the competitive isotope fractionation technique to a wide range of pressing problems in small molecule chemistry and biochemistry.

Inner-Sphere Mechanism for Molecular Oxygen Reduction Catalyzed by Copper Amine Oxidases

Copper and topaquinone (TPQ) containing amine oxidases utilize O2 for the metabolism of biogenic amines while concomitantly generating H2O2 for use by the cell. The mechanism of O2 reduction has been the subject of long-standing debate due to the obscuring influence of a proton-coupled electron transfer between the tyrosine-derived TPQ and copper, a rapidly established equilibrium precluding assignment of the enzyme in its reactive form. Here, we show that substrate-reduced pea seedling amine oxidase (PSAO) exists predominantly in the Cu(I), TPQ semiquinone state. A new mechanistic proposal for O2 reduction is advanced on the basis of thermodynamic considerations together with kinetic studies (at varying pH, temperature, and viscosity), the identification of steady-state intermediates, and the analysis of competitive oxygen kinetic isotope effects, (18)O KIEs, [kcat/KM((16,16)O2)]/[kcat/KM((16,18)O2)]. The (18)O KIE = 1.0136 +/- 0.0013 at pH 7.2 is independent of temperature from 5 degrees C to 47 degrees C and insignificantly changed to 1.0122 +/- 0.0020 upon raising the pH to 9, thus indicating the absence of kinetic complexity. Using density functional methods, the effect is found to be precisely in the range expected for reversible O2 binding to Cu(I) to afford a superoxide, [Cu(II)(eta(1)-O2)(-I)](+), intermediate. Electron transfer from the TPQ semiquinone follows in the first irreversible step to form a peroxide, Cu(II)(eta(1)-O2)(-II), intermediate driving the reduction of O2. The similar (18)O KIEs reported for copper amine oxidases from other sources raise the possibility that all enzymes react by related inner-sphere mechanisms although additional experiments are needed to test this proposal.

The nature of O 2 activation by the ethylene-forming enzyme 1-aminocyclopropane-1-carboxylic acid oxidase

Ethylene is a plant hormone important in many aspects of plant growth and development such as germination, fruit ripening, and senescence. 1-Aminocyclopropane-1-carboxylic acid (ACC) oxidase (ACCO), an O2-activating ascorbate-dependent nonheme iron enzyme, catalyzes the last step in ethylene biosynthesis. The O2 activation process by ACCO was investigated using steady-state kinetics, solvent isotope effects (SIEs), and competitive oxygen kinetic isotope effects (18O KIEs) to provide insights into the nature of the activated oxygen species formed at the active-site iron center and its dependence on ascorbic acid. The observed large 18O KIE of 1.0215 +/- 0.0005 strongly supports a rate-determining step formation of an Fe(IV) O species, which acts as the reactive intermediate in substrate oxidation. The large SIE on kcat/Km(O2) of 5.0 +/- 0.9 suggests that formation of this Fe(IV) O species is linked to a rate-limiting proton or hydrogen atom transfer step. Based on the observed decrease in SIE and 18O KIE values for ACCO at limiting ascorbate concentrations, ascorbate is proposed to bind in a random manner, depending on its concentration. We conclude that ascorbate is not essential for initial O2 binding and activation but is required for rapid Fe(IV) O formation under catalytic turnover. Similar studies can be performed for other nonheme iron enzymes, with the 18O KIEs providing a kinetic probe into the chemical nature of Fe/O2 intermediates formed in the first irreversible step of the O2 activation.

Molecular Oxygen Dependent Steps in Fatty Acid Oxidation by Cyclooxygenase-1

The mechanism by which cyclooxygenase-1 (COX-1), a heme- and tyrosyl radical-containing enzyme, catalyzes the regio- and stereospecific oxygenation of polyunsaturated fatty acids to prostaglandin or hydroperoxide products has not been understood. Steady-state kinetic studies conducted with the native substrate arachidonic acid and the slower substrate linoleic acid are described here. Second-order rate constants, kcat/KM for fatty acid and O2, are found to depend upon the concentration of the other cosubstrate. Competitive oxygen kinetic isotope effects (18O KIEs) kcat/KM(16,16O2)/kcat/KM(18,16O2) reveal that a peroxyl radical is formed in or before the first kinetically irreversible step. Together, the results indicate that the oxygenase reaction occurs by a sequential mechanism which most likely involves reversible abstraction of a hydrogen atom from the fatty acid prior to the trapping of the delocalized substrate radical by O2. The identity of the first kinetically irreversible step, subsequent to forming the peroxyl radical, is also discussed in the context of the magnitude of the oxygen kinetic isotope effects as well as the behavior of kcat/KM(O2) in response to changing solvent pH, pD, and viscosity.

Mechanisms of Electron Transfer in Catalysis by Copper Zinc Superoxide Dismutase

Activated oxygen intermediates during copper zinc superoxide dismutase (SOD) catalysis were investigated using an isotope fractionation technique and natural abundance reagents. Competitive oxygen kinetic isotope effects (KIEs) are reported for the enzyme-catalyzed disproportionation of superoxide as well as the stoichiometric reaction of reduced SOD with molecular oxygen. Analysis within the context of quantum mechanical electron transfer theory provides evidence against an outer-sphere mechanism for O2*- oxidation. A CuII-O2-I intermediate is, therefore, proposed. The SOD-catalyzed oxidation of O2*- is characterized by an inverse (<1) KIE which is similar to those determined for the analogous reactions of synthetic copper compounds. An inverse kinetic isotope effect upon the enzymatic reduction of O2*- is also observed and proposed to arise from rate-determining proton transfer which leads to the formation of HO2* in the SOD active site.

Oxygen Isotope Effects on Electron Transfer to O2Probed Using Chemically Modified Flavins Bound to Glucose Oxidase

Apo-glucose oxidase has been reconstituted with flavins modified in the 7 and 8 positions and characterized with regard to the catalytic rate of O(2) reduction and oxygen-18 isotope effects on this process. Kinetic studies as a function of driving force indicate a reorganization energy for electron transfer to O(2) of lambda = 28 kcal mol(-)(1) at optimal pH, which is similar to the value obtained earlier from temperature dependencies of rates (Roth, J. P.; Klinman, J. P. Proc. Natl. Acad. Sci. U.S.A. 2003, 100, 62-67). For the various enzyme-bound flavins, competitive oxygen-18 kinetic isotope effects fall within the narrow range of 1.0266(5) to 1.0279(6), apparently because of the dominant contribution of outer-sphere reorganization to the activation barrier; within the context of semiclassical and quantum mechanical electron transfer theories, the magnitude of the isotope effects reveals the importance of nuclear tunneling.

Catalysis of electron transfer during activation of O2 by the flavoprotein glucose oxidase.

Two prototropic forms of glucose oxidase undergo aerobic oxidation reactions that convert FADH(-) to FAD and form H(2)O(2) as a product. Limiting rate constants of k(cat)K(M)(O(2)) = (5.7 +/- 1.8) x 10(2) M(-1).s(-1) and k(cat)K(M)(O(2)) = (1.5 +/- 0.3) x 10(6) M(-1).s(-1) are observed at high and low pH, respectively. Reactions exhibit oxygen-18 kinetic isotope effects but no solvent kinetic isotope effects, consistent with mechanisms of rate-limiting electron transfer from flavin to O(2). Site-directed mutagenesis studies reveal that the pH dependence of the rates is caused by protonation of a highly conserved histidine in the active site. Temperature studies (283-323 K) indicate that protonation of His-516 results in a reduction of the activation energy barrier by 6.0 kcal.mol(-1) (0.26 eV). Within the context of Marcus theory, catalysis of electron transfer is attributed to a 19-kcal.mol(-1) (0.82 eV) decrease in the reorganization energy and a much smaller 2.2-kcal.mol(-1) (0.095 eV) enhancement of the reaction driving force. An explanation is advanced that is based on changes in outer-sphere reorganization as a function of pH. The active site is optimized at low pH, but not at high pH or in the H516A mutant where rates resemble the uncatalyzed reaction in solution.

Interpretation of kinetic isotope effects in the decomposition of N 2O over CoO

Nitrogen and oxygen kinetic isotope effects on the decomposition of N 2O on CoO powder, determined experimentally in the temperature range of 575–775 K and at initial N 2O pressures in the range of 40–60 kPa, were interpreted theoretically following the Bigeleisen formalism. Four models of the transition state of the rate determining step were used (linear and bent NNO, cis ONNO, and fork-type NNOO) but only a bent NNO transition state with an interbond angle of 110–140° is consistent with previous experimental data.

Thermal Decomposition of N2O over ZnO: Kinetic Isotope Effects Study

The kinectic parameters were measured for the catalytic decomposition of nitrous oxide on ZnO powder between 40 and 60 kPa and the temperature range 725−835 K. Under studied conditions the reaction was found to be first order in N2O. An Arrhenius fit to the rate constants results in an apparent activation energy of 120 ± 4 kJ mol-1. Only the temperature-dependent nitrogen kinetic isotope effects were experimentally obtained, while the oxygen kinetic isotope effects were perturbed by 18O exchanged between N2O and ZnO. Theoretical interpretation of KIEs was evaluated according to Bigeleisen's formalism. From comparison of calculated results with the experimental data both a cis ONNO and a fork-type NNOO models are acceptable as transition state of the reaction.