New Inhibitors of Laccase and Tyrosinase by Examination of Cross-Inhibition between Copper-Containing Enzymes.

Coppers play crucial roles in the maintenance homeostasis in living species. Approximately 20 enzyme families of eukaryotes and prokaryotes are known to utilize copper atoms for catalytic activities. However, small-molecule inhibitors directly targeting catalytic centers are rare, except for those that act against tyrosinase and dopamine-ß-hydroxylase (DBH). This study tested whether known tyrosinase inhibitors can inhibit the copper-containing enzymes, ceruloplasmin, DBH, and laccase. While most small molecules minimally reduced the activities of ceruloplasmin and DBH, aside from known inhibitors, 5 of 28 tested molecules significantly inhibited the function of laccase, with the Ki values in the range of 15 to 48 µM. Enzyme inhibitory kinetics classified the molecules as competitive inhibitors, whereas differential scanning fluorimetry and fluorescence quenching supported direct bindings. To the best of our knowledge, this is the first report on organic small-molecule inhibitors for laccase. Comparison of tyrosinase and DBH inhibitors using cheminformatics predicted that the presence of thione moiety would suffice to inhibit tyrosinase. Enzyme assays confirmed this prediction, leading to the discovery of two new dual tyrosinase and DBH inhibitors.

Dopamine beta-hydroxylase and its role in regulating the growth and larval metamorphosis in Sinonovacula constricta.

Dopamine beta-hydroxylase (DßH) plays a key role in the synthesis of catecholamines (CAs) in the neuroendocrine regulatory network. The DßH gene was identified from the razor clam Sinonovacula constricta and referred to as ScDßH. The ScDßH gene is a copper type II ascorbate-dependent monooxygenase with a DOMON domain and two Cu2_monooxygen domains. ScDßH transcript expression was abundant in liver and hemolymph. During early development, ScDßH expression significantly increased at the umbo larval stage. Furthermore, the inhibitors and siRNA of DßH were screened. After challenge with DßH inhibitor, the larval metamorphosis and survival rates, and juvenile growth were obviously decreased. Under the siRNA stress, the larval metamorphosis and survival rates were also significantly decreased. Therefore, ScDßH may play an important regulating role in larval metamorphosis and juvenile growth.

Dopamine beta-hydroxylase participate in the immunoendocrine responses of hypothermal stressed white shrimp, Litopenaeus vannamei.

Dopamine beta-hydroxylase (DBH) plays a critical role in catecholamine (CA) synthesis of neuroendocrine regulatory network, and is suggested to be involved in the immunoendocrine responses of invertebrate against bacterial challenge. DBH has been identified in white shrimp, Litopenaeus vannamei, and further investigation on its potential function was conducted after hypothermal stress, pharmaceutical inhibition and gene silencing in the present study. Cloned DBH L. vannamei (LvDBH), belonging to the Copper type II, ascorbate-dependent monooxygenases, was characterized by a DOMON domain, a Cu2_monooxygen domain and three glycosylation sites, and its expression was abundant in thoracic ganglia and haemocytes determined by quantitative real-time PCR. The effects of hypothermal stress showed that LvDBH expression in thoracic ganglia, haemocytes and hepatopancreas as well as the DBH contents in haemocytes and dopamine (DA) and norepinephrine (NE) levels in haemolymph are obviously up-regulated. L. vannamei receiving disulfiram for 30-120 min revealed the inhibition of DBH and NE contents in haemocytes and haemolymph respectively, but high level of DA in haemolymph was noticed. Besides, a significant decrease of LvDBH expression in thoracic ganglia, haemocytes and hepatopancreas were also observed. Subsequently, LvDBH expression was successfully silenced in thoracic ganglia, haemocytes and hepatopancreas of shrimp that received LvDBH-dsRNA for 3 days, and meanwhile, a decrease of DBH contents in haemocytes accompanied by decreased levels of NE and DA in haemolymph were also observed. These results indicate that LvDBH possesses the functional domains responsible for CAs synthesis, and therefore, inhibiting DBH contents in haemocytes by disulfiram and by LvDBH-dsRNA resulted in the impaired synthesis of NE from DA in haemolymph. These also suggest that the increased release of DA and NE in haemolymph for potential modulation of physiological or immunological responses is the consequence of the upregulated LvDBH expression and DBH contents in L. vannamei exposed to hypothermal stress.

Mechanism of O2 activation and substrate hydroxylation in noncoupled binuclear copper monooxygenases.

Peptidylglycine α-hydroxylating monooxygenase (PHM) and dopamine ß-monooxygenase (DßM) are copper-dependent enzymes that are vital for neurotransmitter regulation and hormone biosynthesis. These enzymes feature a unique active site consisting of two spatially separated (by 11 Å in PHM) and magnetically noncoupled copper centers that enables 1e- activation of O2 for hydrogen atom abstraction (HAA) of substrate C-H bonds and subsequent hydroxylation. Although the structures of the resting enzymes are known, details of the hydroxylation mechanism and timing of long-range electron transfer (ET) are not clear. This study presents density-functional calculations of the full reaction coordinate, which demonstrate: (i) the importance of the end-on coordination of superoxide to Cu for HAA along the triplet spin surface; (ii) substrate radical rebound to a CuII hydroperoxide favors the proximal, nonprotonated oxygen; and (iii) long-range ET can only occur at a late step with a large driving force, which serves to inhibit deleterious Fenton chemistry. The large inner-sphere reorganization energy at the ET site is used as a control mechanism to arrest premature ET and dictate the correct timing of ET.

The crystal structure of human dopamine ß-hydroxylase at 2.9 Å resolution.

The norepinephrine pathway is believed to modulate behavioral and physiological processes, such as mood, overall arousal, and attention. Furthermore, abnormalities in the pathway have been linked to numerous diseases, for example hypertension, depression, anxiety, Parkinson's disease, schizophrenia, Alzheimer's disease, attention deficit hyperactivity disorder, and cocaine dependence. We report the crystal structure of human dopamine ß-hydroxylase, which is the enzyme converting dopamine to norepinephrine. The structure of the DOMON (dopamine ß-monooxygenase N-terminal) domain, also found in >1600 other proteins, reveals a possible metal-binding site and a ligand-binding pocket. The catalytic core structure shows two different conformations: an open active site, as also seen in another member of this enzyme family [the peptidylglycine α-hydroxylating (and α-amidating) monooxygenase], and a closed active site structure, in which the two copper-binding sites are only 4 to 5 Å apart, in what might be a coupled binuclear copper site. The dimerization domain adopts a conformation that bears no resemblance to any other known protein structure. The structure provides new molecular insights into the numerous devastating disorders of both physiological and neurological origins associated with the dopamine system.

Characterization of the interaction of the novel antihypertensive etamicastat with human dopamine-ß-hydroxylase: comparison with nepicastat.

The interaction of etamicastat, a novel peripherally acting dopamine-ß-hydroxylase (DBH) inhibitor, with the enzyme was studied using a classical kinetic approach and the pharmacodynamics effect of the compound upon administration to rats was also evaluated. SK-N-SH cell homogenates convert tyramine into octopamine with a Km value of 9 mM, and a Vmax of 1747 nmol/mg protein/h. The K(m) value for ascorbate was 3 mM. The inhibition of DBH by etamicastat and nepicastat, a known centrally acting DBH inhibitor, with IC50 values of 107 and 40 nM, respectively, was fully reversed by dilution. Non-linear fitting of the velocities, determined at various concentrations of substrate (tyramine) and co-substrate (ascorbic acid), and of etamicastat and nepicastat, indicated that the inhibition of DBH by both compounds follows a mixed-model inhibition mechanism, approaching competitive behavior with regards to the substrate tyramine, with K(i) values of 34 and 11 nM, respectively. Relatively to ascorbate, both compounds followed a mixed-model inhibition mechanism, approaching uncompetitive behavior. Oral administration of both compounds (at 30 mg/kg) inhibited adrenal DBH activity over time and significantly decreased noradrenaline levels in the heart. Nepicastat also decreased noradrenaline levels in the parietal cortex, but not etamicastat. Both compounds significantly decreased systolic and diastolic blood pressure in spontaneously hypertensive rats. In conclusion, etamicastat and nepicastat behave as multisubstrate DBH inhibitors, binding reversibly and preferentially to the reduced form of the enzyme, and simultaneously at the substrate and oxygen binding sites. Etamicastat, in contrast to nepicastat, offers the advantage of peripheral selectivity without central effects.

The power of integrating kinetic isotope effects into the formalism of the Michaelis-Menten equation.

The final arbiter of enzyme mechanism is the ability to establish and test a kinetic mechanism. Isotope effects play a major role in expanding the scope and insight derived from the Michaelis-Menten equation. The integration of isotope effects into the formalism of the Michaelis-Menten equation began in the 1970s and has continued until the present. This review discusses a family of eukaryotic copper proteins, including dopamine ß-monooxygenase, tyramine ß-monooxygenase and peptidylglycine α-amidating enzyme, which are responsible for the synthesis of neuroactive compounds, norepinephrine, octopamine and C-terminally carboxamidated peptides, respectively. The review highlights the results of studies showing how combining kinetic isotope effects with initial rate parameters permits the evaluation of: (a) the order of substrate binding to multisubstrate enzymes; (b) the magnitude of individual rate constants in complex, multistep reactions; (c) the identification of chemical intermediates; and (d) the role of nonclassical (tunnelling) behaviour in C-H activation.

Investigation of the hydroxylation mechanism of noncoupled copper oxygenases by ab initio molecular dynamics simulations.

In Nature, the family of copper monooxygenases comprised of peptidylglycine α-hydroxylating monooxygenase (PHM), dopamine ß-monooxygenase (DßM), and tyramine ß-monooxygenase (TßM) is known to perform dioxygen-dependent hydroxylation of aliphatic C-H bonds by using two uncoupled metal sites. In spite of many investigations, including biochemical, chemical, and computational, details of the C-H bond oxygenation mechanism remain elusive. Herein we report an investigation of the mechanism of hydroxylation by PHM by using hybrid quantum/classical potentials (i.e., QM/MM). Although previous investigations using hybrid QM/MM techniques were restricted to geometry optimizations, we have carried out ab initio molecular dynamics simulations in order to include the intrinsic flexibility of the active sites in the modeling protocol. The major finding of this study is an extremely fast rebound step after the initial hydrogen-abstraction step promoted by the cupric-superoxide adduct. The hydrogen-abstraction/rebound sequence leads to the formation of an alkyl hydroperoxide intermediate. Long-range electron transfer from the remote copper site subsequently triggers its reduction to the hydroxylated substrate. We finally show two reactivity consequences inherent in the new mechanistic proposal, the investigation of which would provide a means to check its validity by experimental means.

Active site models for the Cu(A) site of peptidylglycine α-hydroxylating monooxygenase and dopamine ß-monooxygenase.

A mononuclear copper(II) superoxo species has been invoked as the key reactive intermediate in aliphatic substrate hydroxylation by copper monooxygenases such as peptidylglycine α-hydroxylating monooxygenase (PHM), dopamine ß-monooxygenase (DßM), and tyramine ß-monooxygenase (TßM). We have recently developed a mononuclear copper(II) end-on superoxo complex using a N-[2-(2-pyridyl)ethyl]-1,5-diazacyclooctane tridentate ligand, the structure of which is similar to the four-coordinate distorted tetrahedral geometry of the copper-dioxygen adduct found in the oxy-form of PHM (Prigge, S. T.; Eipper, B. A.; Mains, R. E.; Amzel, L. M. Science2004, 304, 864-867). In this study, structures and physicochemical properties as well as reactivity of the copper(I) and copper(II) complexes supported by a series of tridentate ligands having the same N-[2-(2-pyridyl)ethyl]-1,5-diazacyclooctane framework have been examined in detail to shed light on the chemistry dictated in the active sites of mononuclear copper monooxygenases. The ligand exhibits unique feature to stabilize the copper(I) complexes in a T-shape geometry and the copper(II) complexes in a distorted tetrahedral geometry. Low temperature oxygenation of the copper(I) complexes generated the mononuclear copper(II) end-on superoxo complexes, the structure and spin state of which have been further characterized by density functional theory (DFT) calculations. Detailed kinetic analysis on the O(2)-adduct formation reaction gave the kinetic and thermodynamic parameters providing mechanistic insights into the association and dissociation processes of O(2) to the copper complexes. The copper(II) end-on superoxo complex thus generated gradually decomposed to induce aliphatic ligand hydroxylation. Kinetic and DFT studies on the decomposition reaction have suggested that C-H bond abstraction occurs unimolecularly from the superoxo complex with subsequent rebound of the copper hydroperoxo species to generate the oxygenated product. The present results have indicated that a superoxo species having a four-coordinate distorted tetrahedral geometry could be reactive enough to induce the direct C-H bond activation of aliphatic substrates in the enzymatic systems.

Structural insight of dopamine ß-hydroxylase, a drug target for complex traits, and functional significance of exonic single nucleotide polymorphisms.

BACKGROUND: Human dopamine ß-hydroxylase (DBH) is an important therapeutic target for complex traits. Several single nucleotide polymorphisms (SNPs) have also been identified in DBH with potential adverse physiological effect. However, difficulty in obtaining diffractable crystals and lack of a suitable template for modeling the protein has ensured that neither crystallographic three-dimensional structure nor computational model for the enzyme is available to aid rational drug design, prediction of functional significance of SNPs or analytical protein engineering. PRINCIPAL FINDINGS: Adequate biochemical information regarding human DBH, structural coordinates for peptidylglycine alpha-hydroxylating monooxygenase and computational data from a partial model of rat DBH were used along with logical manual intervention in a novel way to build an in silico model of human DBH. The model provides structural insight into the active site, metal coordination, subunit interface, substrate recognition and inhibitor binding. It reveals that DOMON domain potentially promotes tetramerization, while substrate dopamine and a potential therapeutic inhibitor nepicastat are stabilized in the active site through multiple hydrogen bonding. Functional significance of several exonic SNPs could be described from a structural analysis of the model. The model confirms that SNP resulting in Ala318Ser or Leu317Pro mutation may not influence enzyme activity, while Gly482Arg might actually do so being in the proximity of the active site. Arg549Cys may cause abnormal oligomerization through non-native disulfide bond formation. Other SNPs like Glu181, Glu250, Lys239 and Asp290 could potentially inhibit tetramerization thus affecting function. CONCLUSIONS: The first three-dimensional model of full-length human DBH protein was obtained in a novel manner with a set of experimental data as guideline for consistency of in silico prediction. Preliminary physicochemical tests validated the model. The model confirms, rationalizes and provides structural basis for several biochemical data and claims testable hypotheses regarding function. It provides a reasonable template for drug design as well.

A dopamine beta hydroxylase from Chlamys farreri and its induced mRNA expression in the haemocytes after LPS stimulation.

Dopamine beta hydroxylase (DBH) is a critical enzyme in the biosynthesis of catecholamines, and also plays an important role in complex neuroendocrine-immune regulatory network. In the present study, the cDNA encoding dopamine beta hydroxylase (designated CfDBH) was cloned from Chlamys farreri by using rapid amplification of cDNA ends (RACE) approaches and expression sequence tag (EST) analysis. The full-length cDNA of CfDBH was of 2302 bp, containing a 5' untranslated region (UTR) of 32 bp, a 3' UTR of 461 bp with a poly (A) tail, and an open reading frame (ORF) of 1809 bp encoding a polypeptide of 603 amino acids. The deduced amino acid sequence of CfDBH contained a signal peptide, a DOMON domain and a Cu2_monooxygen domain, and it shared 39.4%-42.9% similarity with other reported DBHs. The conserved domains in CfDBH and the amino acid sequence similarity with other DBHs strongly suggested that it was a homologue of DBH in C. farreri. The mRNA expression of CfDBH in various tissues and its temporal expression in haemocytes of scallops stimulated with LPS were ascertained by Quantitative real-time RT-PCR. The mRNA transcripts of CfDBH were detected in all the examined tissues with the highest expression level in hepatopancreas. The expression level of CfDBH in haemocytes was up-regulated after LPS stimulation and increased to hundreds fold higher than that of the control group at 12 h, and then decrease significantly to 0.36-fold and 0.31-fold at 24 h and 48 h respectively. The results suggested pathogen infections significantly induced the expression level of CfDBH, and the activation of DBH could influence the immune response of scallop C. farreri through changing the concentration of catecholamines.

Mononuclear copper(II)-superoxo complexes that mimic the structure and reactivity of the active centers of PHM and DbetaM.

Mononuclear copper(II)-superoxo complexes 2(X)-OO(*) having triplet (S = 1) ground states were obtained via reaction of O(2) with the copper(I) starting materials 1(X) supported by tridentate ligands L(X) [1-(2-p-X-phenethyl)-5-(2-pyridin-2-ylethyl)-1,5-diazacyclooctane; X = CH(3), H, NO(2)] in various solvents. The superoxo complexes 2(X)-OO(*) mimic the structure [tetrahedral geometry with an end-on (eta(1))-bound O(2)(*-)] and the aliphatic C-H bond activation chemistry of peptidylglycine alpha-hydroxylating monooxygenase and dopamine beta-monooxygenase.

N-Hydroxyguanidines oxidation by a N3S copper-complex mimicking the reactivity of Dopamine beta-Hydroxylase.

N-Aryl-N'-hydroxyguanidines are compounds that display interesting pharmacological properties but their chemical reactivity remains poorly investigated. Some of these compounds are substrates for the heme-containing enzymes nitric-oxide synthases (NOS) and act as reducing co-substrates for the copper-containing enzyme Dopamine beta-Hydroxylase (DBH) [P. Slama, J.L. Boucher, M. Réglier, Biochem. Biophys. Res. Commun. 316 (2004) 1081-1087]. DBH catalyses the hydroxylation of the important neurotransmitter dopamine into norepinephrine in the presence of both molecular oxygen and a reducing co-substrate. Although many molecules have been used as co-substrates for DBH, their interaction at the active site of DBH and their role in mechanism are not clearly characterized. In the present paper, we have used a water-soluble copper-N(3)S complex that mimics the Cu(B) site of DBH, and aromatic N-hydroxyguanidines as reducers to address this question. N-Aryl-N'-hydroxyguanidines readily reduced copper(II) to Cu(I) and were oxidized into a nitrosoamidine as previously observed in reactions performed with purified DBH. These data describe for the first time the reactivity of N-aryl-N'-hydroxyguanidines with a water-soluble copper(II) complex and help to understand the interaction of co-substrates with copper at the active site of DBH.

H-atom abstraction reaction for organic substrates via mononuclear copper(II)-superoxo species as a model for DbetaM and PHM.

Hydrogen atom abstraction reactions have been implicated in oxygenation reactions catalyzed by copper monooxygenases such as peptidylglycine alpha-hydroxylating monooxygenase (PHM) and dopamine beta-monooxygenase (DbetaM). We have investigated mononuclear copper(I) and copper(II) complexes with bis[(6-neopentylamino-2-pyridyl)methyl][(2-pyridyl)methyl]amine (BNPA) as functional models for these enzymes. The reaction of [Cu(II)(bnpa)]2+ with H2O2, affords a quasi-stable mononuclear copper(II)-hydroperoxo complex, [Cu(II)(bnpa)(OOH)]+ (4) which is stabilized by hydrophobic interactions and hydrogen bonds in the vicinity of the copper(II) ion. On the other hand, the reaction of [Cu(I)(bnpa)]+ (1) with O2 generates a trans-mu-1,2-peroxo dicopper(II) complex [Cu(II)2(bnpa)2(O2(2-]2+ (2). Interestingly, the same reactions carried out in the presence of exogenous substrates such as TEMPO-H, produce a mononuclear copper(II)-hydroperoxo complex 4. Under these conditions, the H-atom abstraction reaction proceeds via the mononuclear copper(II)-superoxo intermediate [Cu(II)(bnpa)(O2-)]+ (3), as confirmed from indirect observations using a spin trap reagent. Reactions with several substrates having different bond dissociation energies (BDE) indicate that, under our experimental conditions the H-atom abstraction reaction proceeds for substrates with a weak X-H bond (BDE < 72.6 kcal mol(-1)). These investigations indicate that the copper(II)-hydroperoxo complex is a useful tool for elucidation of H-atom abstraction reaction mechanisms for exogenous substrates. The useful functionality of the complex has been achieved via careful control of experimental conditions and the choice of appropriate ligands for the complex.

Mechanism of the insect enzyme, tyramine beta-monooxygenase, reveals differences from the mammalian enzyme, dopamine beta-monooxygenase.

Tyramine beta-monooxygenase (TbetaM) catalyzes the synthesis of the neurotransmitter, octopamine, in insects. Kinetic and isotope effect studies have been carried out to determine the kinetic mechanism of TbetaM for comparison with the homologous mammalian enzymes, dopamine beta-monooxygenase and peptidylglycine alpha-hydroxylating monooxygenase. A new and distinctive feature of TbetaM is very strong substrate inhibition that is dependent on the level of the co-substrate, O(2), and reductant as well as substrate deuteration. This has led to a model in which tyramine can bind to either the Cu(I) or Cu(II) forms of TbetaM, with substrate inhibition ameliorated at very high ascorbate levels. The rate of ascorbate reduction of the E-Cu(II) form of TbetaM is also reduced at high tyramine, leading us to propose the existence of a binding site for ascorbate to this class of enzymes. These findings may be relevant to the control of octopamine production in insect cells.

The DOMON domains are involved in heme and sugar recognition.

UNLABELLED: We expand the functionally uncharacterized DOMON domain superfamily to identify several novel families, including the first prokaryotic representatives. Using several computational tools we show that it is involved in ligand binding--either as heme- or sugar-binding domains. We present evidence that the DOMON domain along with the DM13 domain comprises a novel electron-transfer system potentially involved in oxidative modification of animal cell-surface proteins. Other novel versions might function as sugar sensors of histidine kinases of bacterial two component systems. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online and also at ftp://ftp.ncbi.nih.gov/pub/aravind/domon/.

Advances in studying bioinorganic reaction mechanisms: isotopic probes of activated oxygen intermediates in metalloenzymes.

Metalloenzymes catalyze reactions of molecular oxygen and its reduced forms through the controlled formation of metal-bound, activated oxygen intermediates. These intermediates have been a challenge to characterize and new experimental approaches capable of relating structure to reactivity under physiologically relevant conditions are needed. The application of a competitive isotope fractionation technique has enabled changes in O-O bonding to be probed during enzyme-catalyzed reactions. The derived isotope effects provide insights into the reaction mechanisms of O2 and O2*-, which probably could not have been obtained using more conventional methods.

Thioether sulfur oxygenation from O2 or H2O2 reactivity of copper complexes with tridentate N2Sthioether ligands.

To model thioether-copper coordination chemistry including oxidative reactivity, such as occurs in the copper monooxygenases peptidylglycine -hydroxylating monooxygenase (PHM) and dopamine beta-hydroxylase (DbetaH), we have synthesized new tridentate N2S ligands LSEP and LSBz [LSEP = methyl(2-phenethylsulfanylpropyl)(2-pyridin-2-ylethyl)amine; LSBz = (2-benzylsulfanylpropyl)methyl(2-pyridin-2-ylethyl)amine)]. Both copper(I) and copper(II) complexes have been prepared, and their respective O2 and H2O2 chemistry has been studied. Under mild conditions, oxygenation of [(LSEP)CuI]+ (1a) and [(LSBz)CuI]+ (2a) leads to ligand sulfoxidation, thus exhibiting copper monooxygenase activity. A copper(II) complex of this sulfoxide ligand product, [(LSOEP)CuII(CH3OH)(OClO3)2], has been structurally characterized, demonstrating Cu-Osulfoxide ligation. The X-ray structure of [(LSEP)CuII(H2O)(OClO3)]+ (1b) and its solution UV-visible spectral properties [S-CuII LMCT band at 365 nm (MeCN solvent); epsilon = 4285 M-1 cm-1] indicate the thioether sulfur atom is bound to the cupric ion in both the solid (CuII-S distance: 2.31 A) and solution states. Reaction of 1b with H2O2 leads to sulfonation via the sulfoxide; excess hydrogen peroxide gives mostly sulfone product. These results may provide some insight into recent reports concerning protein methionine oxidation, showing the potential importance of copper-mediated oxidation processes in certain biological settings.

Characterization of the structure and reactivity of monocopper-oxygen complexes supported by beta-diketiminate and anilido-imine ligands.

Copper-oxygen complexes supported by beta-diketiminate and anilido-imine ligands have recently been reported (Aboelella et al., J Am Chem Soc 2004, 126, 16896; Reynolds et al., Inorg Chem 2005, 44, 6989) as potential biomimetic models for dopamine beta-monooxygenase (DbetaM) and peptidylglycine alpha-hydroxylating monooxygenase (PHM). However, in contrast to the enzymatic systems, these complexes fail to exhibit C--H hydroxylation activity (Reynolds et al., Chem Commun 2005, 2014). Quantum chemical characterization of the 1:1 Cu-O(2) model adducts and related species (Cu(III)-hydroperoxide, Cu(III)-oxo, and Cu(III)-hydroxide) indicates that the 1:1 Cu-O(2) adducts are unreactive toward substrates because of the weakness of the O--H bond that would be formed upon hydrogen-atom abstraction. This in turn is ascribed to the 1:1 adducts having both low reduction potentials and basicities. Cu(III)-oxo species on the other hand, determined to be intermediate between Cu(III)-oxo and Cu(II)-oxyl in character, are shown to be far more reactive toward substrates. Based on these results, design strategies for new DbetaM and PHM biomimetic ligands are proposed: new ligands should be made less electron rich so as to favor end-on dioxygen coordination in the 1:1 Cu-O(2) adducts. Comparison of the relative reactivities of the various copper-oxygen complexes as hydroxylating agents provides support for a Cu(II)-superoxide species as the intermediate responsible for substrate hydroxylation in DbetaM and PHM, and suggests that a Cu(III)-oxo intermediate would be competent in this process as well.

Catalytic mechanism of dopamine beta-monooxygenase mediated by Cu(III)-oxo.

Mechanisms of dopamine hydroxylation by the Cu(II)-superoxo species and the Cu(III)-oxo species of dopamine beta-monooxygenase (DBM) are discussed using QM/MM calculations for a whole-enzyme model of 4700 atoms. A calculated activation barrier for the hydrogen-atom abstraction by the Cu(II)-superoxo species is 23.1 kcal/mol, while that of the Cu(III)-oxo, which can be viewed as Cu(II)-O*, is 5.4 kcal/mol. Energies of the optimized radical intermediate in the superoxo- and oxo-mediated pathways are 18.4 and -14.2 kcal/mol, relative to the corresponding reactant complexes, respectively. These results demonstrate that the Cu(III)-oxo species can better mediate dopamine hydroxylation in the protein environment of DBM. The side chains of three amino acid residues (His415, His417, and Met490) coordinate to the Cu(B) atom, one of the copper sites in the catalytic core that plays a role for the catalytic function. The hydrogen-bonding network between dopamine and the three amino acid residues (Glu268, Glu369, and Tyr494) plays an essential role in substrate binding and the stereospecific hydroxylation of dopamine to norepinephrine. The dopamine hydroxylation by the Cu(III)-oxo species is a downhill and lower-barrier process toward the product direction with the aid of the protein environment of DBM. This enzyme is likely to use the high reactivity of the Cu(III)-oxo species to activate the benzylic C-H bond of dopamine; the enzymatic reaction can be explained by the so-called oxygen rebound mechanism.