Competitive oxygen kinetic isotope effects (18O KIEs) on water oxidation initiated by ruthenium oxo (RuO) complexes are examined here as a means to formulate mechanisms of O–O bond formation, which is a critical step in the production of “solar hydrogen”. The kinetics of three structurally related catalysts are investigated to complement the measurement and computation of 18O KIEs, derived from the analysis of O2 relative to natural abundance H2O under single and multi-turnover conditions. The findings reported here support and extend mechanistic proposals from 18O tracer studies conducted exclusively under non-catalytic conditions. It is shown how density functional theory calculations, when performed in tandem with experiments, can constrain mechanisms of catalytic water oxidation and help discriminate between them.

Heme oxygenase is responsible for the degradation of a histidine-ligated ferric protoporphyrin IX (Por) to biliverdin, CO, and the free ferrous ion. Described here are studies of tyrosyl radical formation reactions that occur after oxidizing FeIII(Por) to FeIV=O(Por·+) in human heme oxygenase isoform-1 (hHO-1) and the structurally homologous protein from Corynebacterium diphtheriae (cdHO). Site-directed mutagenesis on hHO-1 probes the reduction of FeIV=O(Por·+) by tyrosine residues within 11 Å of the prosthetic group. In hHO-1, Y58· is implicated as the most likely site of oxidation, based on the pH and pD dependent kinetics. The absence of solvent deuterium isotope effects in basic solutions of hHO-1 and cdHO contrasts with the behavior of these proteins in the acidic solution, suggesting that long-range proton-coupled electron transfer predominates over electron transfer.

The mechanism of O2 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 O2 to H2O2. Steady-state kinetics indicate that amine-reduced CuAGAO is reoxidized by O2 >103 times faster than the CoAGAO analogue. Complementary spectroscopic studies reveal that the difference in the second order rate constant, kcat/KM(O2), arises from the more negative redox potential of CoIII/II in relation to CuII/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 (η1)-metal bound superoxide is reduced to an η1-hydroperoxide in the rate-limiting step.

Molecular oxygen is produced from water via the following reaction of potassium ferrate (K2FeO4) in acidic solution: 4[H3FeVIO4]+ + 8H3O+ → 4Fe3+ + 3O2 + 18H2O. This study focuses upon the mechanism by which the O–O bond is formed. Stopped-flow kinetics at variable acidities in H2O and D2O are used to complement the analysis of competitive oxygen-18 kinetic isotope effects (18O KIEs) upon consumption of natural abundance water. The derived 18O 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 18O 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 18O KIEs.

Oxygen isotope fractionation is applied for the first time to probe the catalytic oxidation of water using a widely studied ruthenium complex, [RuII(tpy)(bpy)(H2O)](ClO4)2 (bpy = 2,2′-bipyridine; tpy = 2,2′;6″,2″-terpyridine). Competitive oxygen-18 kinetic isotope effects (18O KIEs) derived from the ratio of 16,16O2 to 16,18O2 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 18O KIE exhibits the greatest degree of O–O coupling in the transition state. Smaller 18O 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.

Cyclooxygenases-1 and -2 are tyrosyl radical (Y·)-utilizing hemoproteins responsible for the biosynthesis of lipid-derived autocoids. COX-2, in particular, is a primary mediator of inflammation and believed to be up-regulated in many forms of cancer. Described here are first-of-a-kind studies of COX-2-catalyzed oxidation of the substrate analogue linoleic acid. Very large (≥20) temperature-independent deuterium kinetic isotope effects (KIEs) on the rate constant for enzyme turnover were observed, due to hydrogen atom abstraction from the bisallylic C–H(D) of the fatty acid. The magnitude of the KIE depends on the O2 concentration, consistent with reversible H/D tunneling mediated by the catalytic Y·. At physiological levels of O2, retention of the hydrogen initially abstracted by the catalytic tyrosine results in strongly temperature-dependent KIEs on O–H(D) homolysis, also characteristic of nuclear tunneling.

Rice α-(di)oxygenase mediates the regio- and stereospecific oxidation of fatty acids using a persistent catalytic tyrosyl radical. Experiments conducted in the physiological O2 concentration range, where initial hydrogen atom abstraction from the fatty acid occurs in a kinetically reversible manner, are described. Our findings indicate that O2-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.

The steady-state catalytic mechanism of a fatty acid α-(di)oxygenase is examined, revealing that a persistent tyrosyl radical (Tyr379•) effects O2 insertion into Cα−H bonds of fatty acids. The initiating Cα−H homolysis step is characterized by apparent rate constants and deuterium kinetic isotope effects (KIEs) that increase hyperbolically upon raising the concentration of O2. These results are consistent with H• tunneling, transitioning from a reversible to an irreversible regime. The limiting deuterium KIEs increase from ∼30 to 120 as the fatty acid chain is shortened from that of the native substrate. In addition, activation barriers increase in a manner that reflects decreased fatty acid binding affinities. Anaerobic isotope exchange experiments provide compelling evidence that Tyr379• initiates catalysis by H• abstraction. Cα−H homolysis is kinetically driven by O2 trapping of the α-carbon radical and reduction of a putative peroxyl radical intermediate to a 2(R)-hydroperoxide product. These findings add to a body of work which establishes large-scale hydrogen tunneling in proteins. This particular example is novel because it involves a protein-derived amino acid radical.

Oxidative transformations using molecular oxygen are widespread in nature but remain a major challenge in chemical synthesis. Limited mechanistic understanding presents the main obstacle to exploiting O2 in “bioinspired” industrial processes. Isotopic methods are presently being applied to characterize reactions of natural abundance O2 including its coordination to reduced transition metals and cleavage of the O−O bond. This review describes the application of competitive oxygen-18 isotope effects, together with Density Functional Theory, to examine O2 reductive activation under catalytically relevant conditions. The approach should be generally useful for probing small-molecule activation by transition-metal complexes.

Competitively determined oxygen (18O) 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 (18O KIEs) and equilibrium isotope effects (18O 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 18O EIEs. Researchers in mechanistic biological inorganic chemistry would like to differentiate “inner-sphere” from “outer-sphere” reactivity of O2, 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 18O KIEs to 18O 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.

Oxygen equilibrium isotope effects (18O EIEs) upon the formation of metal superoxide and peroxide structures from natural abundance O2 are reported. The 18O EIEs determined over a range of temperatures are compared to those calculated on the basis of vibrational frequencies. Considering all vibrational modes in a “full frequency model” is found to reproduce the empirical results better than “cut-off” models which consider only the most isotopically sensitive modes. Theoretically, the full frequency model predicts that 18O EIEs arise from competing enthalpic and entropic influences resulting in nonlinear variations with temperature. Experimental evidence is provided for an increase in the magnitude of the EIE, in some instances implicating a change from inverse to normal values, as the temperature is raised. This finding is not easily reconciled with the common intuition that 18O EIEs arise from a reduction of the O−O force constant and attendant changes in zero point energy level splitting. Instead a dominant entropic effect, as described here, is expected to characterize isotope effects upon reversible binding of small molecules to metal centers in enzymes and inorganic compounds.

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 CuI, 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, 18O KIEs, [kcat/KM(16,16O2)]/[kcat/KM(16,18O2)]. The 18O KIE = 1.0136 ± 0.0013 at pH 7.2 is independent of temperature from 5 °C to 47 °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 CuI to afford a superoxide, [CuII(η1-O2)−I]+, intermediate. Electron transfer from the TPQ semiquinone follows in the first irreversible step to form a peroxide, CuII(η1-O2)−II, intermediate driving the reduction of O2. The similar 18O 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.

Competitive oxygen kinetic isotope effects (18O KIEs) on water oxidation initiated by ruthenium oxo (RuO) complexes are examined here as a means to formulate mechanisms of O–O bond formation, which is a critical step in the production of “solar hydrogen”. The kinetics of three structurally related catalysts are investigated to complement the measurement and computation of 18O KIEs, derived from the analysis of O2 relative to natural abundance H2O under single and multi-turnover conditions. The findings reported here support and extend mechanistic proposals from 18O tracer studies conducted exclusively under non-catalytic conditions. It is shown how density functional theory calculations, when performed in tandem with experiments, can constrain mechanisms of catalytic water oxidation and help discriminate between them.