Kinetic basis for the activation of human cyclooxygenase-2 rather than cyclooxygenase-1 by nitric oxide.

Numerous studies have shown that nitric oxide (NO) interacts with human cyclooxygenase (COX); however, conflicting results exist with respect to their interactions. Herein, recombinant human COX-1 and COX-2 were prepared and treated with NO donors individually under anaerobic and aerobic conditions. The S-nitrosylation detection and subsequent kinetic investigations into the arachidonic acid (AA) oxidation of COX enzymes indicate that NO S-nitrosylates both COX-1 and COX-2 in an oxygen-dependent manner, but enhances only the dioxygenase activity of COX-2. The solution viscosity, deuterium kinetic isotope effect (KIE), and oxygen-18 KIE experiments further demonstrate that NO activates COX-2 by altering the protein conformation to stimulate substrate association/product release and by accelerating the rate of hydrogen abstraction from AA by catalytic tyrosine radicals. These novel findings provide useful information for designing new drugs with less cardiotoxic effects that can block the interaction between NO and COX.

Submolecular regulation of cell transformation by deuterium depleting water exchange reactions in the tricarboxylic acid substrate cycle.

The naturally occurring isotope of hydrogen ((1)H), deuterium ((2)H), could have an important biological role. Deuterium depleted water delays tumor progression in mice, dogs, cats and humans. Hydratase enzymes of the tricarboxylic acid (TCA) cycle control cell growth and deplete deuterium from redox cofactors, fatty acids and DNA, which undergo hydride ion and hydrogen atom transfer reactions. A model is proposed that emphasizes the terminal complex of mitochondrial electron transport chain reducing molecular oxygen to deuterium depleted water (DDW); this affects gluconeogenesis as well as fatty acid oxidation. In the former, the DDW is thought to diminish the deuteration of sugar-phosphates in the DNA backbone, helping to preserve stability of hydrogen bond networks, possibly protecting against aneuploidy and resisting strand breaks, occurring upon exposure to radiation and certain anticancer chemotherapeutics. DDW is proposed here to link cancer prevention and treatment using natural ketogenic diets, low deuterium drinking water, as well as DDW production as the mitochondrial downstream mechanism of targeted anti-cancer drugs such as Avastin and Glivec. The role of (2)H in biology is a potential missing link to the elusive cancer puzzle seemingly correlated with cancer epidemiology in western populations as a result of excessive (2)H loading from processed carbohydrate intake in place of natural fat consumption.

A Revised Mechanism for Human Cyclooxygenase-2.

The mechanism of ω-6 polyunsaturated fatty acid oxidation by wild-type cyclooxygenase 2 and the Y334F variant, lacking a conserved hydrogen bond to the catalytic tyrosyl radical/tyrosine, was examined for the first time under physiologically relevant conditions. The enzymes show apparent bimolecular rate constants and deuterium kinetic isotope effects that increase in proportion to co-substrate concentrations before converging to limiting values. The trends exclude multiple dioxygenase mechanisms as well as the proposal that initial hydrogen atom abstraction from the fatty acid is the first irreversible step in catalysis. Temperature dependent kinetic studies reinforce the novel finding that hydrogen transfer from the reduced catalytic tyrosine to a terminal peroxyl radical is the first irreversible step that controls regio- and stereospecific product formation.

Tyrosine oxidation in heme oxygenase: examination of long-range proton-coupled electron transfer.

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 Fe(III)(Por) to Fe(IV)=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 Fe(IV)=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.

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.

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.

Hydrogen tunneling steps in cyclooxygenase-2 catalysis.

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 O(2) concentration, consistent with reversible H/D tunneling mediated by the catalytic Y·. At physiological levels of O(2), 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.

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.

Catalytic mechanism of a heme and tyrosyl radical-containing fatty acid α-(di)oxygenase.

The steady-state catalytic mechanism of a fatty acid α-(di)oxygenase is examined, revealing that a persistent tyrosyl radical (Tyr379(•)) effects O(2) insertion into C(α)-H bonds of fatty acids. The initiating C(α)-H homolysis step is characterized by apparent rate constants and deuterium kinetic isotope effects (KIEs) that increase hyperbolically upon raising the concentration of O(2). 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. C(α)-H homolysis is kinetically driven by O(2) 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.

Oxygen isotope effects as structural and mechanistic probes in inorganic oxidation chemistry.

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 O(2) in "bioinspired" industrial processes. Isotopic methods are presently being applied to characterize reactions of natural abundance O(2) 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 O(2) reductive activation under catalytically relevant conditions. The approach should be generally useful for probing small-molecule activation by transition-metal complexes.

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.

Computational modeling of oxygen isotope effects on metal-mediated O2 activation at varying temperatures.

Oxygen equilibrium isotope effects ((18)O EIEs) upon the formation of metal superoxide and peroxide structures from natural abundance O(2) are reported. The (18)O 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 (18)O 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 (18)O 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.

Evidence for protein radical-mediated nuclear tunneling in fatty acid alpha-oxygenase.

Rice alpha-oxygenase (RalphaO) catalyzes the insertion of O2 into the Calpha-H bond of various fatty acids. The mechanism is thought to involve a tyrosyl radical as the oxidant on the basis of comparisons to the structurally homologous cyclooxygenase enzymes. Kinetic and spectroscopic results presented here for the wild-type RalphaO and the Tyr379Phe mutant indicate an irreversible H* abstraction mechanism and support the involvement of the proposed catalytic Tyr*. In addition, very large, weakly temperature dependent deuterium kinetic isotope effects (approximately 50) are observed, consistent with extensive nuclear tunneling. RalphaO, thus, presents a novel example where such quantum effects are associated with an amino acid radical-utilizing enzyme.

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.

Direct examination of H2O2 activation by a heme peroxidase.

Described here is the application of oxygen isotope fractionation together with computational methods, to elucidate a mechanism of enzymatic H2O2 activation. Horseradish peroxidase (HRP) has been the subject of intensive experimental and computational studies, yet questions remain as to the reversibility of the O-O cleavage step. New insight is afforded by the competitive oxygen kinetic isotope effect (18O KIE) upon H2O2 consumption determined under turnover conditions. The 18O KIE is compared to isotope effects calculated for the O-O heterolysis transition state and potential intermediates using density functional theory. In addition, experiments in enriched water provide evidence for HRP-catalyzed scrambling of the 18O label into the unreacted H2O2. The results provide an unprecedented view of H2O2 activation by a heme peroxidase and challenge the assumption of rate-limiting O-O heterolysis.

The first crystal structure of a monomeric phenoxyl radical: 2,4,6-tri-tert-butylphenoxyl radical.

Crystals of the 2,4,6-tri-tert-butylphenoxyl radical have been isolated and characterized by X-ray diffraction, and calculations have been performed that give the distribution of spin density in the radical.

Isotopic probing of molecular oxygen activation at copper(I) sites.

Copper-dioxygen (CuO2) adducts are frequently proposed as intermediates in enzymes, yet their electronic and vibrational structures have not always been understood. [Cu(eta1-O2)TMG3tren]+ (TMG3tren = 1,1,1-tris{2-[N2-(1,1,3,3-tetramethylguanidino)]ethyl}amine) features end-on (eta1) O2 coordination in the solid state. Described here is an investigation of the compound's solution properties by nuclear magnetic resonance spectroscopy, density functional calculations, and oxygen isotope effects. The study yields two major findings. First, [Cu(eta1-O2)TMG3tren]+ is paramagnetic due to a triplet electronic structure; this is in contrast to other copper compounds where O2 is bound in a side-on manner. Second, the oxygen equilibrium isotope effect upon O2 binding to copper(I) (18O EIE [triple bond] K(16O16O)/K(16O18O) = 1.0148 +/- 0.0012) is significantly larger than those determined for iron and cobalt eta1-O2 adducts. This result is suggested to reflect greater ionic (CuII-O2-I) character within the valence bond description. A revised interpretation of the physical origins of the 18O EIEs upon O2 binding to redox metals is also advanced along with experimental data that should be used as benchmarks for interpreting 18O kinetic isotope effects upon enzyme reactions.

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.