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.     

A theoretical study of the mechanism for the biogenesis of cofactor topaquinone in copper amine oxidases

In the present quantum chemical study, the biogenesis of the cofactor topaquinone (TPQ) has been studied using hybrid density functional theory (B3LYP). The suggested mechanism is divided into six steps and incorporates the observation of four crystallized intermediates. The experimental suggestion that the formation of the Cu(II)-peroxy species is the rate-limiting step is consistent with the results of the present study. Before the formation of the Cu(II)-peroxy species, dioxygen is suggested to first bind at the equatorial position on the copper metal center. A mechanism for the critical O-O bond cleavage is suggested, and this step is found to be driven by an unusually large exothermicity. A complex, spin-forbidden formation of H(2)O(2) with and without the involvement of the copper metal center is discussed. The results are discussed in detail, and comparisons are made to experimental findings and suggestions.     

Trapping of a dopaquinone intermediate in the TPQ cofactor biogenesis in a copper-containing amine oxidase from Arthrobacter globiformis

The biogenesis of the topaquinone (TPQ) cofactor of copper amine oxidase (CAO) is self-catalyzed and requires copper and molecular oxygen. A dopaquinone intermediate has been proposed to undergo 1,4-addition of a copper-associated water molecule to form the reduced form of TPQ (TPQ(red)), followed by facile oxidation by O(2) to yield the mature TPQ (TPQ(ox)). In this study, we have incorporated a lysine residue in the active site of Arthrobacter globiformis CAO (AGAO) by site-directed mutagenesis to produce D298K-AGAO. The X-ray crystal structure of D298K-AGAO at 1.7-A resolution revealed that a covalent linkage formed between the epsilon-amino side chain of Lys298 and the C2 position of a dopaquinone derived from Tyr382, a precursor to TPQ(ox). We assigned the species as an iminoquinone tautomer (LTI) of lysine tyrosylquinone (LTQ), the organic cofactor of lysyl oxidase (LOX). The time course of the formation of LTI at pH 6.8 was followed by UV/vis and resonance Raman spectroscopies. In the early phase of the reaction, an LTQ-like intermediate was observed. This intermediate then slowly converted to LTI in an isosbestic manner. Not only is the presence of a dopaquinone intermediate in the TPQ biogenesis confirmed, but it also provides strong support for the proposed intermediacy of a dopaquinone in the biogenesis of LTQ in LOX. Further, this study indicates that the dopaquinone intermediate in AGAO is mobile and can swing from the copper site into the active-site wedge to react with Lys298.     

Quinone‐Catalyzed Selective Oxidation of Organic Molecules

Quinones are common stoichiometric reagents in organic chemistry. Para‐quinones with high reduction potentials, such as DDQ and chloranil, are widely used and typically promote hydride abstraction. In recent years, many catalytic applications of these methods have been achieved by using transition metals, electrochemistry, or O₂ to regenerate the oxidized quinone in situ. Complementary studies have led to the development of a different class of quinones that resemble the ortho‐quinone cofactors in copper amine oxidases and mediate the efficient and selective aerobic and/or electrochemical dehydrogenation of amines. The latter reactions typically proceed by electrophilic transamination and/or addition‐elimination reaction mechanisms, rather than hydride abstraction pathways. The collective observations show that the quinone structure has a significant influence on the reaction mechanism and has important implications for the development of new quinone reagents and quinone‐catalyzed transformations.     

Enantiomer-specific binding of ruthenium(II) molecular wires by the amine oxidase of Arthrobacter globiformis

The copper amine oxidase from Arthrobacter globiformis (AGAO) is reversibly inhibited by molecular wires comprising a Ru(II) complex head group and an aromatic tail group joined by an alkane linker. The crystal structures of a series of Ru(II)-wire-AGAO complexes differing with respect to the length of the alkane linker have been determined. All wires lie in the AGAO active-site channel, with their aromatic tail group in contact with the trihydroxyphenylalanine quinone (TPQ) cofactor of the enzyme. The TPQ cofactor is consistently in its active ("off-Cu") conformation, and the side chain of the so-called "gate" residue Tyr296 is consistently in the "gate-open" conformation. Among the wires tested, the most stable complex is produced when the wire has a -(CH2)4- linker. In this complex, the Ru(II)(phen)(bpy)2 head group is level with the protein molecular surface. Crystal structures of AGAO in complex with optically pure forms of the C4 wire show that the linker and head group in the two enantiomers occupy slightly different positions in the active-site channel. Both the Lambda and Delta isomers are effective competitive inhibitors of amine oxidation. Remarkably, inhibition by the C4 wire shows a high degree of selectivity for AGAO in comparison with other copper-containing amine oxidases.     

Detailed chemomechanistic sensitivity study on the alkoxylation of fatty amines

The mechanism of the autocatalytic alkoxylation of fatty amines was elucidated using a combined experimental and theoretical approach. The kinetic parameters of the elementary reaction steps are fitted to the experimental data gained in semibatch for propylene and butylene oxides with dodecylamine. A quality-of-fit sensitivity study was conducted to assess the robustness and accuracy of the model. Herein, we identified the kinetic parameters that are either crucial for a good fit or rate controlling in terms of the overall kinetics. It was found that the critical steps are the activation of the epoxide ring by a hydroxyl group present on either the intermediate or final product, highlighting the autocatalytic nature of the reaction. Furthermore, we introduced a so-called degree of conversion control to characterize the importance of each elementary reaction step toward the epoxide conversion. The degree of conversion control showed that the uncatalyzed route toward the secondary amine is only influential at low conversion. At higher conversion the route via the catalytic intermediate dominates. Alternative mechanisms were investigated as well but did not significantly improve the quality of fitting and were thus discarded.     

3-pyrrolines are mechanism-based inactivators of the quinone-dependent amine oxidases but only substrates of the flavin-dependent amine oxidases

We previously reported that 3-pyrroline and 3-phenyl-3-pyrroline effect a time-dependent inactivation of the copper-containing quinone-dependent amine oxidase from bovine plasma (BPAO) (Lee et al. J. Am. Chem. Soc. 1996, 118, 7241-7242). Quinone cofactor model studies suggested a mechanism involving stoichiometric turnover to a stable pyrrolylated cofactor. Full details of the model studies are now reported along with data on the inhibition of BPAO by a family of 3-aryl-3-pyrrolines (aryl = substituted phenyl, 1-naphthyl, 2-naphthyl), with the 4-methoxy-3-nitrophenyl analogue being the most potent. At the same time, the parent 3-phenyl analogue is a pure substrate for the flavin-dependent mitochondrial monoamine oxidase B from bovine liver. Spectroscopic studies (including resonance Raman) on BPAO inactivated by the 4-methoxy-3-nitrophenyl analogue are consistent with covalent derivatization of the 2,4,5-trihydroxyphenylalanine quinone (TPQ) cofactor. The distinction of a class of compounds acting as an inactivator of one amine oxidase family and a pure substrate of another amine oxidase family represents a unique lead to the development of selective inhibitors of the mammalian copper-containing amine oxidases.     

A dopaquinone model that mimics the water addition step of cofactor biogenesis in copper amine oxidases

The consensus mechanism for biogenesis of the 2,4,5-trihydroxyphenylalanine quinone (TPQ) cofactor in copper amine oxidases involves a key water addition to the dopaquinone intermediate. Although hydration of o-quinones seems straightforward and was implicated previously in aqueous autoxidation of catechols to give ultimately hydroxyquinones, a recent study (Mandal, S.; Lee, Y.; Purdy, M. M.; Sayre, L. M. J. Am. Chem. Soc. 2000, 122, 3574-3584) showed that the observed hydroxyquinones arise not from hydration, but from addition to the o-quinones of H(2)O(2) generated during autoxidation of the catechols. In the enzyme case, hydration of dopaquinone is proposed to be mediated by the active site Cu(II). To establish precedent for this mechanism, we engineered a catechol tethered to a Cu(II)-coordinating unit, such that the corresponding o-quinone could be generated in situ by oxidation with periodate (to avoid generation of H(2)O(2)). Thus, coordination of 4-((2-(bis(2-pyridylmethyl)amino)ethylamino)methyl)-1,2-benzenediol (1) to Cu(II) and subsequent addition of periodate resulted in rapid formation of the TPQ-like corresponding hydroxyquinone. Hydroxyquinone formation was seen also using Zn(II) and Ni(II), but not in the absence of M(II). Under the same conditions, periodate oxidation of the simple catechol 4-tert-butylcatechol does not give hydroxyquinone in the presence or absence of Cu(II). M(II)OH(2) pK(a) data for the Cu(II), Zn(II), and Ni(II) complexes with the pendant tetradentate ligand in the masked (dimethyl ether) catechol form, and kinetic pH-rate profiles of the metal-dependent hydroxyquinone formation from periodate oxidation of catechol 1, suggested a rate-limiting addition step of the ligand-coordinated M(II)OH to the o-quinone intermediate. This study represents the first chemical demonstration of a true o-quinone hydration, which occurs in cofactor biogenesis in copper amine oxidases.     

Reactions of copper(II)-phenol systems with O2: models for TPQ biosynthesis in copper amine oxidases

Copper(II) complexes supported by a series of phenol-containing bis(pyridin-2-ylmethyl)amine N(3) ligands (denoted as L(o)H, L(m)H, and L(p)H) have been synthesized, and their O(2) reactivity has been examined in detail to gain mechanistic insights into the biosynthesis of the TPQ cofactor (2,4,5-trihydroxyphenylalaninequinone, TOPA quinone) in copper-containing amine oxidases. The copper(II) complex of L(o)H (ortho-phenol derivative) involves a direct phenolate to copper(II) coordination and exhibits almost no reactivity toward O(2) at 60 °C in CH(3)OH. On the other hand, the copper(II) complex of L(m)H (meta-phenol derivative), which does not involve direct coordinative interaction between the phenol moiety and the copper(II) ion, reacts with O(2) in the presence of triethylamine as a base to give a methoxy-substituted para-quinone derivative under the same conditions. The product structure has been established by detailed nuclear magnetic resonance (NMR), infrared (IR) spectroscopy, and electrospray ionization-mass spectroscopy (ESI-MS) (including (18)O-labeling experiment) analyses. Density functional theory predicts that the reaction involves (i) intramolecular electron transfer from the deprotonated phenol (phenolate) to copper(II) to generate a copper(I)-phenoxyl radical; (ii) the addition of O(2) to this intermediate, resulting in an end-on copper(II) superoxide; (iii) electrophilic substitution of the phenolic radical to give a copper(II)-alkylperoxo intermediate; (iv) O-O bond cleavage concomitant with a proton migration, giving a para-quinone derivative; and (v) Michael addition of methoxide from copper(II) to the para-quinone ring and subsequent O(2) oxidation. This reaction sequence is similar to that proposed for the biosynthetic pathway leading to the TPQ cofactor in the enzymatic system. The generated para-quinone derivative can act as a turnover catalyst for aerobic oxidation of benzylamine to N-benzylidene benzylamine. Another type of copper(II)-phenol complex with an L(p)H ligand (para-phenol derivative) also reacts with O(2) under the same experimental conditions. However, the product of this reaction is a keto-alcohol derivative, the structure of which is qualitatively different from that of the cofactor. These results unambiguously demonstrate that the steric relationship between the phenol moiety and the supported copper(II) ion is decisive in the conversion of active-site tyrosine residues to the TPQ cofactor.     

Catalytic turnover of benzylamine by a model for the lysine tyrosylquinone (LTQ) cofactor of lysyl oxidase

Lysyl oxidase differs from other copper amine oxidases in that its active quinone cofactor reflects cross-linking of a lysyl residue into the tyrosine-derived quinone nucleus found in the plasma and other copper amine oxidases. A model for the lysyl oxidase cofactor (LTQ), 3,3-dimethyl-2,3-dihydroindole-5,6-quinone (4), was synthesized and found to be stable to both hydrolysis and oxidation events that prevent simpler models from functioning as turnover catalysts. We show that 4 catalyzes the aerobic oxidative deamination of benzylamine, though turnover eventually ceases on account of oxidation of the dihydrobenzoxazole tautomer of the "product Schiff base" to form a benzoxazole, a reaction that may be physiologically relevant. The mechanism of the overall reaction profile was elucidated by a combination of optical and NMR spectroscopy and O(2) uptake studies.     

Role of copper ion in bacterial copper amine oxidase: spectroscopic and crystallographic studies of metal-substituted enzymes

The role of the active site Cu(2+) of phenylethylamine oxidase from Arthrobacter globiformis (AGAO) has been studied by substitution with other divalent cations, where we were able to remove >99.5% of Cu(2+) from the active site. The enzymes reconstituted with Co(2+) and Ni(2+) (Co- and Ni-AGAO) exhibited 2.2 and 0.9% activities, respectively, of the original Cu(2+)-enzyme (Cu-AGAO), but their K(m) values for amine substrate and dioxygen were comparable. X-ray crystal structures of the Co- and Ni-AGAO were solved at 2.0-1.8 A resolution. These structures revealed changes in the metal coordination environment when compared to that of Cu-AGAO. However, the hydrogen-bonding network around the active site involving metal-coordinating and noncoordinating water molecules was preserved. Upon anaerobic mixing of the Cu-, Co-, and Ni-AGAO with amine substrate, the 480 nm absorption band characteristic of the oxidized form of the topaquinone cofactor (TPQ(ox)) disappeared rapidly (< 6 ms), yielding the aminoresorcinol form of the reduced cofactor (TPQ(amr)). In contrast to the substrate-reduced Cu-AGAO, the semiquinone radical (TPQ(sq)) was not detected in Co- and Ni-AGAO. Further, in the latter, TPQ(amr) reacted reversibly with the product aldehyde to form a species with a lambda(max) at around 350 nm that was assigned as the neutral form of the product Schiff base (TPQ(pim)). Introduction of dioxygen to the substrate-reduced Co- and Ni-AGAO resulted in the formation of a TPQ-related intermediate absorbing at around 360 nm, which was assigned to the neutral iminoquinone form of the 2e(-)-oxidized cofactor (TPQ(imq)) and which decayed concomitantly with the generation of TPQ(ox). The rate of TPQ(imq) formation and its subsequent decay in Co- and Ni-AGAO was slow when compared to those of the corresponding reactions in Cu-AGAO. The low catalytic activities of the metal-substituted enzymes are due to the impaired efficiencies of the oxidative half-reaction in the catalytic cycle of amine oxidation. On the basis of these results, we propose that the native Cu(2+) ion has essential roles such as catalyzing the electron transfer between TPQ(amr) and dioxygen, in part by providing a binding site for 1e(-)- and 2e(-)-reduced dioxygen species to be efficiently protonated and released and also preventing the back reaction between the product aldehyde and TPQ(amr).     

A comparison between artificial and natural water oxidation

Two artificial water oxidation catalysts, the blue dimer and the Llobet catalyst, have been studied using hybrid DFT methods. The results are compared to those for water oxidation in the natural photosystem II enzyme. Studies on the latter system have now reached a high level of understanding, at present much higher than the one for the artificial systems. A recent high resolution X-ray structural investigation of PSII has confirmed the main features of the structure of the oxygen evolving complex (OEC) suggested by previous DFT cluster studies. The O-O bond formation mechanism suggested is of direct coupling (DC) type between an oxygen radical and a bridging oxo ligand. A similar DC mechanism is found for the Llobet catalyst, while an acid-base (AB) mechanism is preferred for the blue dimer. All of them require at least one oxygen radical. Full energy diagrams, including both redox and chemical steps, have been constructed illustrating similarities and differences to the natural system. Unlike previous DFT studies, the results of the present study suggest that the blue dimer is rate-limited by the initial redox steps, and the Llobet catalyst by O(2) release. The results could be useful for further improvement of the artificial systems.     

A combined quantum and molecular mechanical study of the O2 reductive cleavage in the catalytic cycle of multicopper oxidases

The four-electron reduction of dioxygen to water in multicopper oxidases takes place in a trinuclear copper cluster, which is linked to a mononuclear blue copper site, where the substrates are oxidized. Recently, several intermediates in the catalytic cycle have been spectroscopically characterized, and two possible structural models have been suggested for both the peroxy and native intermediates. In this study, these spectroscopic results are complemented by hybrid quantum and molecular mechanical (QM/MM) calculations, taking advantage of recently available crystal structures with a full complement of copper ions. Thereby, we obtain optimized molecular structures for all of the experimentally studied intermediates involved in the reductive cleavage of the O(2) molecule and energy profiles for individual reaction steps. This allows identification of the experimentally observed intermediates and further insight into the reaction mechanism that is probably relevant for the whole class of multicopper oxidases. We suggest that the peroxy intermediate contains an O(2)(2-) ion, in which one oxygen atom bridges the type 2 copper ion and one of the type 3 copper ions, whereas the other one coordinates to the other type 3 copper ion. One-electron reduction of this intermediate triggers the cleavage of the O-O bond, which involves the uptake of a proton. The product of this cleavage is the observed native intermediate, which we suggest to contain a O(2-) ion coordinated to all three of the copper ions in the center of the cluster.     

Tailoring D‐Amino Acid Oxidase from the Pig Kidney to R‐Stereoselective Amine Oxidase and its Use in the Deracemization of α‐Methylbenzylamine

The deracemization of racemic amines to yield enantioenriched amines using S‐stereoselective amine oxidases (AOx) has recently been attracting attention. However, R‐stereoselective AOx that are suitable for deracemization have not yet been identified. An R‐stereoselective AOx was now evolved from porcine kidney D ‐amino acid oxidase (pkDAO) and subsequently use for the deracemization of racemic amines. The engineered pkDAO, which was obtained by directed evolution, displayed a markedly changed substrate specificity towards R amines. The mutant enzyme exhibited a high preference towards the substrate α‐methylbenzylamine and was used to synthesize the S amine through deracemization. The findings of this study indicate that further investigations on the structure–activity relationship of AOx are warranted and also provide a new method for biotransformations in organic synthesis.     

A comparative computational investigation on the proton and hydride transfer mechanisms of monoamine oxidase using model molecules

Monoamine oxidase (MAO) enzymes regulate the level of neurotransmitters by catalyzing the oxidation of various amine neurotransmitters, such as serotonin, dopamine and norepinephrine. Therefore, they are the important targets for drugs used in the treatment of depression, Parkinson, Alzeimer and other neurodegenerative disorders. Elucidation of MAO-catalyzed amine oxidation will provide new insights into the design of more effective drugs. Various amine oxidation mechanisms have been proposed for MAO so far, such as single electron transfer mechanism, polar nucleophilic mechanism and hydride mechanism. Since amine oxidation reaction of MAO takes place between cofactor flavin and the amine substrate, we focus on the small model structures mimicking flavin and amine substrates so that three model structures were employed. Reactants, transition states and products of the polar nucleophilic (proton transfer), the water-assisted proton transfer and the hydride transfer mechanisms were fully optimized employing various semi-empirical, ab initio and new generation density functional theory (DFT) methods. Activation energy barriers related to these mechanisms revealed that hydride transfer mechanism is more feasible.     

仿生合成中的单核铜蛋白及其含铜金属模拟酶的研究

仿生化学的研究重点之一是对各类天然金属酶的氧化模拟。文章主要综述了近年来国内外对含金属铜的单核蛋白质体蓝素结构、半乳糖氧化酶、含铜的胺氧化酶和铜锌超氧歧化酶等的结构特点和催化功能的研究进展。质体蓝素结构为畸变的四面体,参与光合作用过程中电子传递;半乳糖氧化酶活性中心结构是平面四边形,能将伯醇氧化成相应的醛;铜锌超氧歧化...     

Mechanistic Study on Rh‐Catalyzed Stereoselective CC/CH Activation of tert‐Cyclobutanols

A mechanistic study was performed on the Rh‐catalyzed stereoselective C?C/C?H activation of tert‐cyclobutanols. The present study corroborated the previous proposal that the reaction occurs by metalation, β‐C elimination, 1,4‐Rh transfer, C?O insertion, and a final catalyst‐regeneration step. The rate‐determining step was found to be the 1,4‐Rh transfer step, whereas the stereoselectivity‐determining step did not correspond to any of the aforementioned steps. It was found that both the thermodynamic stability of the product of the β‐C elimination and the kinetic feasibility of the 1,4‐Rh transfer and C?O insertion steps made important contributions. In other words, three steps (i.e., β‐C elimination, 1,4‐Rh transfer, and C?O insertion) were found to be important in determining the configurations of the two quaternary stereocenters.     

Is there computational support for an unprotonated carbon in the E4 state of nitrogenase?

In the key enzyme for nitrogen fixation in nature, nitrogenase, the active site has a metal cluster with seven irons and one molybdenum bound by bridging sulfurs. Surprisingly, there is also a carbon in the center of the cluster, with a role that is not known. A mechanism has been suggested experimentally, where two hydrides leave as a hydrogen molecule in the critical E₄ state. A structure with two hydrides, two protonated sulfurs and an unprotonated carbon has been suggested for this state. Rather recently, DFT calculations supported the experimental mechanism but found an active state where the central carbon is protonated all the way to CH₃. Even more recently, another DFT study was made that instead supported the experimentally suggested structure. To sort out the origin of these quite different computational results, additional calculations have here been performed using different DFT functionals. The conclusion from these calculations is very clear and shows no computational support for an unprotonated carbon in E₄. © 2017 Wiley Periodicals, Inc.     

Dimerization of HNO in aqueous solution: an interplay of solvation effects, fast acid-base equilibria, and intramolecular hydrogen bonding?

The recent unraveling of the rather complex acid-base equilibrium of nitroxyl (HNO) has stimulated a renewed interest in the significance of HNO for biology and pharmacy. HNO plays an important role in enzymatic mechanisms and is discussed as a potential therapeutic agent against heart failure. A cumbersome property for studying HNO reactions, its fast dimerization leading to the rapid formation of N(2)O, is surprisingly far from being well understood. It prevents isolation and limits intermediate concentrations of nitroxyl in solution. In this study, a new mechanism for the HNO dimerization reaction in aqueous solution has been theoretically derived on the basis of DFT calculations. Detailed analysis of the initial reaction step suggests a reversal of the cis-trans isomer preference in solution compared to the corresponding gas phase reaction. In contrast to a gas phase derived model based on intramolecular rearrangement steps, an acid-base equilibrium model is in agreement with previous experimental findings and, moreover, explains the fundamental differences between the well studied gas phase reaction and the solvent reaction in terms of polarity, cis-trans isomerizations, and acidities of the intermediates. In the case of cis-hyponitrous acid, the calculated pK(a) values of the acid-base equilibria were found to be significantly different from the corresponding experimental value of the stable trans isomer. Under physiological conditions, N(2)O formation is dominated by the decomposition of the unstable monoanion cis-N(2)O(2)H(-) rather than that of the commonly stated cis-HONNOH.