Justine Roth - Academia.edu (original) (raw)
Papers by Justine Roth
Geochmica et Cosmochimica Acta
Journal of biological inorganic chemistry : JBIC : a publication of the Society of Biological Inorganic Chemistry, 2014
Heme oxygenase is responsible for the degradation of a histidine-ligated ferric protoporphyrin IX... more 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.
Analytical Chemistry, 2014
The reduction chemistry of molecular oxygen underpins the energy metabolism of multicellular orga... more 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.
The Journal of Physical Chemistry A, 2009
Oxygen equilibrium isotope effects ((18)O EIEs) upon the formation of metal superoxide and peroxi... more 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.
Science, 2001
The transfer of a hydrogen atom-a proton and an electron-is a fundamental process in chemistry an... more The transfer of a hydrogen atom-a proton and an electron-is a fundamental process in chemistry and biology. A variety of hydrogen atom transfer reactions, involving iron complexes, phenols, hydroxylamines, tBuOOH, toluene, and related radicals, are shown to follow the Marcus cross relation. Thus, the Marcus theory formalism based on ground-state energetics and self-exchange rates, originally developed for electron transfer processes, is also valuable for hydrogen atom transfer. Compounds that undergo slow proton transfer (C-H bonds) or slow electron transfer (cobalt complexes) also undergo slow hydrogen atom transfer. Limitations of this approach are also discussed.
Proceedings of the National Academy of Sciences, 2003
Two prototropic forms of glucose oxidase undergo aerobic oxidation reactions that convert FADH(-)... more 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.
Journal of the American Chemical Society, 2006
JA066369R . Left: 18 O fractionation due to oxidation of (Cu I ,Zn)SOD by O2; data are fitted to ... more JA066369R . Left: 18 O fractionation due to oxidation of (Cu I ,Zn)SOD by O2; data are fitted to Rf/R0 ) f (1/KIEap-1) . 8b Right: simulation of 18 O KIEs as a function of ∆G°for outer-sphere ET to O2 (see text for details).
Journal of the American Chemical Society, 2011
Cyclooxygenases-1 and -2 are tyrosyl radical (Y 3 )-utilizing hemoproteins responsible for the bi... more Cyclooxygenases-1 and -2 are tyrosyl radical (Y 3 )-utilizing hemoproteins responsible for the biosynthesis of lipid-derived autocoids. COX-2, in particular, is a primary mediator of inflammation and believed to be upregulated in many forms of cancer. Described here are firstof-a-kind studies of COX-2-catalyzed oxidation of the substrate analogue linoleic acid. Very large (g20) temperatureindependent deuterium kinetic isotope effects (KIEs) on the rate constant for enzyme turnover were observed, due to hydrogen atom abstraction from the bisallylic CÀH(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 3 . At physiological levels of O 2 , retention of the hydrogen initially abstracted by the catalytic tyrosine results in strongly temperature-dependent KIEs on OÀH(D) homolysis, also characteristic of nuclear tunneling.
Journal of the American Chemical Society, 2006
Identifying intermediates in catalytic oxidation reactions requires the development of new probes... more Identifying intermediates in catalytic oxidation reactions requires the development of new probes of structure and mechanism. Reported here are proof-of-concept studies of oxygen (18O) isotope effects upon reversible O2-binding reactions of classic inorganic compounds. It is shown that the 18O equilibrium isotope effects may be used to differentiate structures where O2 is bound as a side-on peroxide ligand versus an end-on superoxide ligand. The application of 18O equilibrium isotope effects to the interpretation of 18O kinetic isotope effects and the study of O2 activation mechanisms is also discussed.
Journal of the American Chemical Society, 2000
Self-exchange reactions between high-spin iron complexes of 2,2′-bi-imidazoline (H 2 bim) have be... more Self-exchange reactions between high-spin iron complexes of 2,2′-bi-imidazoline (H 2 bim) have been investigated by the dynamic NMR line-broadening technique. Addition of the ferric complex [Fe III (H 2 bim) 3 ] 3+ causes broadening of the 1 H NMR resonances of the ferrous analogue, [Fe II (H 2 bim) 3 ] 2+ . This indicates electron self-exchange with k e -) (1.7 ( 0.2) × 10 4 M -1 s -1 at 298 K in MeCN-d 3 (µ ) 0.1 M). Similar broadening is observed when the deprotonated ferric complex [Fe III (Hbim)(H 2 bim) 2 ] 2+ is added to [Fe II (H 2 bim) 3 ] 2+ . Because these reactants differ by a proton and an electron, this is a net hydrogen atom exchange reaction. Kinetic and thermodynamic results preclude stepwise mechanisms of sequential proton and then electron transfer, or electron and then proton transfer. Concomitant electron and proton (H • ) transfer occurs with bimolecular rate constant k H • ) (5.8 ( 0.6) × 10 3 M -1 s -1 . This is a factor of 3 smaller than k e -under the same conditions. The H-atom exchange reaction exhibits a primary kinetic isotope effect k NH /k ND ) 2.3 ( 0.3 at 324 K, whereas no such effect is detected in the electron exchange reaction. Proton self-exchange between the two ferric complexes, [Fe III (Hbim)(H 2 bim) 2 ] 2+ and [Fe III (H 2 bim) 3 ] 3+ , has also been investigated and is found to be faster than both the electron and H-atom transfer reactions. From kinetic analyses and the application of simple Marcus theory, an order of intrinsic reaction barriers λ H • > λ e -> λ H + is derived. The reorganization energies are discussed in terms of their inner-sphere and outer-sphere components.
Journal of the American Chemical Society, 2005
Glucose oxidase mediates the aerobic oxidation of simple sugars to lactones using a noncovalently... more Glucose oxidase mediates the aerobic oxidation of simple sugars to lactones using a noncovalently bound flavin cofactor. The chemical mechanism of this reaction has been uncertain for many years. Here it is shown, using enzymes reconstituted with chemically modified cofactors, that sugar oxidation most likely occurs by concerted hydride (H-) abstraction. Studies of the kinetics and thermodynamics together with the application of Marcus theory reveal a large reorganization energy barrier. The magnitude of this intrinsic contribution appears characteristic of H- transfer in proteins and in solution. The observation that neither the thermodynamics nor reorganization energy is significantly altered in the glucose oxidase active site raises questions concerning how the redox reaction may be catalyzed.
Journal of the American Chemical Society, 2008
Journal of the American Chemical Society, 2008
Figure 3. Determination of D kcat/KM by GC/MS analysis. The resolution of d 29 and h29 pentadecan... more Figure 3. Determination of D kcat/KM by GC/MS analysis. The resolution of d 29 and h29 pentadecanal at (a) 100% and (b) ∼1% product formation.
Journal of the American Chemical Society, 2007
Copper-dioxygen (CuO2) adducts are frequently proposed as intermediates in enzymes, yet their ele... more Copper-dioxygen (CuO2) adducts are frequently proposed as intermediates in enzymes, yet their electronic and vibrational structures have not always been understood. [Cu(η 1 -O2)TMG3tren] + (TMG3tren ) 1,1,1-tris{2-[N 2 -(1,1,3,3-tetramethylguanidino)]ethyl}amine) features end-on (η 1 ) 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(η 1 -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) ( 18 O EIE ≡ K( 16 O 16 O)/K( 16 O 18 O) ) 1.0148 ( 0.0012) is significantly larger than those determined for iron and cobalt η 1 -O2 adducts. This result is suggested to reflect greater ionic (Cu II -O2 -I ) character within the valence bond description. A revised interpretation of the physical origins of the 18 O EIEs upon O2 binding to redox metals is also advanced along with experimental data that should be used as benchmarks for interpreting 18 O kinetic isotope effects upon enzyme reactions.
Journal of the American Chemical Society, 2005
Apo-glucose oxidase has been reconstituted with flavins modified in the 7 and 8 positions and cha... more 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.
Journal of the American Chemical Society, 2003
Reported here are self-exchange reactions between iron 2,2&am... more Reported here are self-exchange reactions between iron 2,2'-bi(tetrahydro)pyrimidine (H(2)bip) complexes and between cobalt 2,2'-biimidazoline (H(2)bim) complexes. The (1)H NMR resonances of [Fe(II)(H(2)bip)(3)](2+) are broadened upon addition of [Fe(III)(H(2)bip)(3)](3+), indicating that electron self-exchange occurs with k(Fe,e)(-) = (1.1 +/- 0.2) x 10(5) M(-1) s(-1) at 298 K in CD(3)CN. Similar studies of [Fe(II)(H(2)bip)(3)](2+) plus [Fe(III)(Hbip)(H(2)bip)(2)](2+) indicate that hydrogen-atom self-exchange (proton-coupled electron transfer) occurs with k(Fe,H.) = (1.1 +/- 0.2) x 10(4) M(-1) s(-1) under the same conditions. Both self-exchange reactions are faster at lower temperatures, showing small negative enthalpies of activation: DeltaH++(e(-)) = -2.1 +/- 0.5 kcal mol(-1) (288-320 K) and DeltaH++(H.) = -1.5 +/- 0.5 kcal mol(-1) (260-300 K). This behavior is concluded to be due to the faster reaction of the low-spin states of the iron complexes, which are depopulated as the temperature is raised. Below about 290 K, rate constants for electron self-exchange show the more normal decrease with temperature. There is a modest kinetic isotope effect on H-atom self-exchange of 1.6 +/- 0.5 at 298 K that is close to that seen previously for the fully high-spin iron biimidazoline complexes.(12) The difference in the measured activation parameters, E(a)(D) - E(a)(H), is -1.2 +/- 0.8 kcal mol(-1), appears to be inconsistent with a semiclassical view of the isotope effect, and suggests extensive tunneling. Reactions of [Co(H(2)bim)(3)](2+)-d(24) with [Co(H(2)bim)(3)](3+) or [Co(Hbim)(H(2)bim)(2)](2+) occur with scrambling of ligands indicating inner-sphere processes. The self-exchange rate constant for outer-sphere electron transfer between [Co(H(2)bim)(3)](2+) and [Co(H(2)bim)(3)](3+) is estimated to be 10(-)(6) M(-1) s(-1) by application of the Marcus cross relation. Similar application of the cross relation to H-atom transfer reactions indicates that self-exchange between [Co(H(2)bim)(3)](2+) and [Co(Hbim)(H(2)bim)(2)](2+) is also slow, < or =10(-3) M(-1) s(-1). The slow self-exchange rates for the cobalt complexes are apparently due to their interconverting high-spin [Co(II)(H(2)bim)(3)](2+) with low-spin Co(III) derivatives.
Journal of the American Chemical Society, 2005
We report a mechanistically based study of bifunctional catalyst systems in which chiral nucleoph... more We report a mechanistically based study of bifunctional catalyst systems in which chiral nucleophiles work in conjunction with Lewis acids to produce -lactams in high chemical yield, diastereoselectivity, and enantioselectivity. Chiral cinchona alkaloid derivatives work best when paired with Lewis acids based on Al(III), Zn(II), Sc(III), and, most notably, In(III). Homogeneous bifunctional catalysts, in which the catalyst contains both Lewis acidic and Lewis basic sites, were also studied in detail. Mechanistic evidence allows us to conclude that the chiral nucleophiles form zwitterionic enolates that react with metalcoordinated imines. Alternative scenarios, which postulated metal-bound enolates, were disfavored on the basis of our observations.
Geochmica et Cosmochimica Acta
Journal of biological inorganic chemistry : JBIC : a publication of the Society of Biological Inorganic Chemistry, 2014
Heme oxygenase is responsible for the degradation of a histidine-ligated ferric protoporphyrin IX... more 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.
Analytical Chemistry, 2014
The reduction chemistry of molecular oxygen underpins the energy metabolism of multicellular orga... more 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.
The Journal of Physical Chemistry A, 2009
Oxygen equilibrium isotope effects ((18)O EIEs) upon the formation of metal superoxide and peroxi... more 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.
Science, 2001
The transfer of a hydrogen atom-a proton and an electron-is a fundamental process in chemistry an... more The transfer of a hydrogen atom-a proton and an electron-is a fundamental process in chemistry and biology. A variety of hydrogen atom transfer reactions, involving iron complexes, phenols, hydroxylamines, tBuOOH, toluene, and related radicals, are shown to follow the Marcus cross relation. Thus, the Marcus theory formalism based on ground-state energetics and self-exchange rates, originally developed for electron transfer processes, is also valuable for hydrogen atom transfer. Compounds that undergo slow proton transfer (C-H bonds) or slow electron transfer (cobalt complexes) also undergo slow hydrogen atom transfer. Limitations of this approach are also discussed.
Proceedings of the National Academy of Sciences, 2003
Two prototropic forms of glucose oxidase undergo aerobic oxidation reactions that convert FADH(-)... more 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.
Journal of the American Chemical Society, 2006
JA066369R . Left: 18 O fractionation due to oxidation of (Cu I ,Zn)SOD by O2; data are fitted to ... more JA066369R . Left: 18 O fractionation due to oxidation of (Cu I ,Zn)SOD by O2; data are fitted to Rf/R0 ) f (1/KIEap-1) . 8b Right: simulation of 18 O KIEs as a function of ∆G°for outer-sphere ET to O2 (see text for details).
Journal of the American Chemical Society, 2011
Cyclooxygenases-1 and -2 are tyrosyl radical (Y 3 )-utilizing hemoproteins responsible for the bi... more Cyclooxygenases-1 and -2 are tyrosyl radical (Y 3 )-utilizing hemoproteins responsible for the biosynthesis of lipid-derived autocoids. COX-2, in particular, is a primary mediator of inflammation and believed to be upregulated in many forms of cancer. Described here are firstof-a-kind studies of COX-2-catalyzed oxidation of the substrate analogue linoleic acid. Very large (g20) temperatureindependent deuterium kinetic isotope effects (KIEs) on the rate constant for enzyme turnover were observed, due to hydrogen atom abstraction from the bisallylic CÀH(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 3 . At physiological levels of O 2 , retention of the hydrogen initially abstracted by the catalytic tyrosine results in strongly temperature-dependent KIEs on OÀH(D) homolysis, also characteristic of nuclear tunneling.
Journal of the American Chemical Society, 2006
Identifying intermediates in catalytic oxidation reactions requires the development of new probes... more Identifying intermediates in catalytic oxidation reactions requires the development of new probes of structure and mechanism. Reported here are proof-of-concept studies of oxygen (18O) isotope effects upon reversible O2-binding reactions of classic inorganic compounds. It is shown that the 18O equilibrium isotope effects may be used to differentiate structures where O2 is bound as a side-on peroxide ligand versus an end-on superoxide ligand. The application of 18O equilibrium isotope effects to the interpretation of 18O kinetic isotope effects and the study of O2 activation mechanisms is also discussed.
Journal of the American Chemical Society, 2000
Self-exchange reactions between high-spin iron complexes of 2,2′-bi-imidazoline (H 2 bim) have be... more Self-exchange reactions between high-spin iron complexes of 2,2′-bi-imidazoline (H 2 bim) have been investigated by the dynamic NMR line-broadening technique. Addition of the ferric complex [Fe III (H 2 bim) 3 ] 3+ causes broadening of the 1 H NMR resonances of the ferrous analogue, [Fe II (H 2 bim) 3 ] 2+ . This indicates electron self-exchange with k e -) (1.7 ( 0.2) × 10 4 M -1 s -1 at 298 K in MeCN-d 3 (µ ) 0.1 M). Similar broadening is observed when the deprotonated ferric complex [Fe III (Hbim)(H 2 bim) 2 ] 2+ is added to [Fe II (H 2 bim) 3 ] 2+ . Because these reactants differ by a proton and an electron, this is a net hydrogen atom exchange reaction. Kinetic and thermodynamic results preclude stepwise mechanisms of sequential proton and then electron transfer, or electron and then proton transfer. Concomitant electron and proton (H • ) transfer occurs with bimolecular rate constant k H • ) (5.8 ( 0.6) × 10 3 M -1 s -1 . This is a factor of 3 smaller than k e -under the same conditions. The H-atom exchange reaction exhibits a primary kinetic isotope effect k NH /k ND ) 2.3 ( 0.3 at 324 K, whereas no such effect is detected in the electron exchange reaction. Proton self-exchange between the two ferric complexes, [Fe III (Hbim)(H 2 bim) 2 ] 2+ and [Fe III (H 2 bim) 3 ] 3+ , has also been investigated and is found to be faster than both the electron and H-atom transfer reactions. From kinetic analyses and the application of simple Marcus theory, an order of intrinsic reaction barriers λ H • > λ e -> λ H + is derived. The reorganization energies are discussed in terms of their inner-sphere and outer-sphere components.
Journal of the American Chemical Society, 2005
Glucose oxidase mediates the aerobic oxidation of simple sugars to lactones using a noncovalently... more Glucose oxidase mediates the aerobic oxidation of simple sugars to lactones using a noncovalently bound flavin cofactor. The chemical mechanism of this reaction has been uncertain for many years. Here it is shown, using enzymes reconstituted with chemically modified cofactors, that sugar oxidation most likely occurs by concerted hydride (H-) abstraction. Studies of the kinetics and thermodynamics together with the application of Marcus theory reveal a large reorganization energy barrier. The magnitude of this intrinsic contribution appears characteristic of H- transfer in proteins and in solution. The observation that neither the thermodynamics nor reorganization energy is significantly altered in the glucose oxidase active site raises questions concerning how the redox reaction may be catalyzed.
Journal of the American Chemical Society, 2008
Journal of the American Chemical Society, 2008
Figure 3. Determination of D kcat/KM by GC/MS analysis. The resolution of d 29 and h29 pentadecan... more Figure 3. Determination of D kcat/KM by GC/MS analysis. The resolution of d 29 and h29 pentadecanal at (a) 100% and (b) ∼1% product formation.
Journal of the American Chemical Society, 2007
Copper-dioxygen (CuO2) adducts are frequently proposed as intermediates in enzymes, yet their ele... more Copper-dioxygen (CuO2) adducts are frequently proposed as intermediates in enzymes, yet their electronic and vibrational structures have not always been understood. [Cu(η 1 -O2)TMG3tren] + (TMG3tren ) 1,1,1-tris{2-[N 2 -(1,1,3,3-tetramethylguanidino)]ethyl}amine) features end-on (η 1 ) 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(η 1 -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) ( 18 O EIE ≡ K( 16 O 16 O)/K( 16 O 18 O) ) 1.0148 ( 0.0012) is significantly larger than those determined for iron and cobalt η 1 -O2 adducts. This result is suggested to reflect greater ionic (Cu II -O2 -I ) character within the valence bond description. A revised interpretation of the physical origins of the 18 O EIEs upon O2 binding to redox metals is also advanced along with experimental data that should be used as benchmarks for interpreting 18 O kinetic isotope effects upon enzyme reactions.
Journal of the American Chemical Society, 2005
Apo-glucose oxidase has been reconstituted with flavins modified in the 7 and 8 positions and cha... more 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.
Journal of the American Chemical Society, 2003
Reported here are self-exchange reactions between iron 2,2&am... more Reported here are self-exchange reactions between iron 2,2'-bi(tetrahydro)pyrimidine (H(2)bip) complexes and between cobalt 2,2'-biimidazoline (H(2)bim) complexes. The (1)H NMR resonances of [Fe(II)(H(2)bip)(3)](2+) are broadened upon addition of [Fe(III)(H(2)bip)(3)](3+), indicating that electron self-exchange occurs with k(Fe,e)(-) = (1.1 +/- 0.2) x 10(5) M(-1) s(-1) at 298 K in CD(3)CN. Similar studies of [Fe(II)(H(2)bip)(3)](2+) plus [Fe(III)(Hbip)(H(2)bip)(2)](2+) indicate that hydrogen-atom self-exchange (proton-coupled electron transfer) occurs with k(Fe,H.) = (1.1 +/- 0.2) x 10(4) M(-1) s(-1) under the same conditions. Both self-exchange reactions are faster at lower temperatures, showing small negative enthalpies of activation: DeltaH++(e(-)) = -2.1 +/- 0.5 kcal mol(-1) (288-320 K) and DeltaH++(H.) = -1.5 +/- 0.5 kcal mol(-1) (260-300 K). This behavior is concluded to be due to the faster reaction of the low-spin states of the iron complexes, which are depopulated as the temperature is raised. Below about 290 K, rate constants for electron self-exchange show the more normal decrease with temperature. There is a modest kinetic isotope effect on H-atom self-exchange of 1.6 +/- 0.5 at 298 K that is close to that seen previously for the fully high-spin iron biimidazoline complexes.(12) The difference in the measured activation parameters, E(a)(D) - E(a)(H), is -1.2 +/- 0.8 kcal mol(-1), appears to be inconsistent with a semiclassical view of the isotope effect, and suggests extensive tunneling. Reactions of [Co(H(2)bim)(3)](2+)-d(24) with [Co(H(2)bim)(3)](3+) or [Co(Hbim)(H(2)bim)(2)](2+) occur with scrambling of ligands indicating inner-sphere processes. The self-exchange rate constant for outer-sphere electron transfer between [Co(H(2)bim)(3)](2+) and [Co(H(2)bim)(3)](3+) is estimated to be 10(-)(6) M(-1) s(-1) by application of the Marcus cross relation. Similar application of the cross relation to H-atom transfer reactions indicates that self-exchange between [Co(H(2)bim)(3)](2+) and [Co(Hbim)(H(2)bim)(2)](2+) is also slow, < or =10(-3) M(-1) s(-1). The slow self-exchange rates for the cobalt complexes are apparently due to their interconverting high-spin [Co(II)(H(2)bim)(3)](2+) with low-spin Co(III) derivatives.
Journal of the American Chemical Society, 2005
We report a mechanistically based study of bifunctional catalyst systems in which chiral nucleoph... more We report a mechanistically based study of bifunctional catalyst systems in which chiral nucleophiles work in conjunction with Lewis acids to produce -lactams in high chemical yield, diastereoselectivity, and enantioselectivity. Chiral cinchona alkaloid derivatives work best when paired with Lewis acids based on Al(III), Zn(II), Sc(III), and, most notably, In(III). Homogeneous bifunctional catalysts, in which the catalyst contains both Lewis acidic and Lewis basic sites, were also studied in detail. Mechanistic evidence allows us to conclude that the chiral nucleophiles form zwitterionic enolates that react with metalcoordinated imines. Alternative scenarios, which postulated metal-bound enolates, were disfavored on the basis of our observations.