Nature of the intermediate formed in the reduction of O(2) to H(2)O at the trinuclear copper cluster active site in native laccase

The multicopper oxidases contain at least four copper atoms and catalyze the four-electron reduction of O(2) to H(2)O at a trinuclear copper cluster. An intermediate, termed native intermediate, has been trapped by a rapid freeze-quench technique from Rhus vernicifera laccase when the fully reduced form reacts with dioxygen. This intermediate had been described as an oxygen-radical bound to the trinuclear copper cluster with one Cu site reduced. XAS, however, shows that all copper atoms are oxidized in this intermediate. A combination of EXAFS, multifrequency EPR, and VTVH MCD has been used to understand how this fully oxidized trinuclear Cu cluster relates to the fully oxidized resting form of the enzyme. It is determined that in the native intermediate all copper atoms of the cluster are bridged by the product of full O(2) reduction. In contrast, the resting form has one copper atom of the cluster (the T2 Cu) magnetically isolated from the others. The native intermediate decays to the resting oxidized form with a rate that is too slow to be in the catalytic cycle. Thus, the native intermediate appears to be the catalytically relevant fully oxidized form of the enzyme, and its role in catalysis is considered.     

Electrochemical Studies of Intramolecular Electron Transfer in Laccase from Trametes versicolor

This paper describes electrochemical behavior of laccase from the fungus Trametes versicolor. The issues related to discrimination of the redox potentials corresponding to copper centers T1 and T2/T3 in the active site and possible mechanism of intramolecular electron transfer have been discussed. The electron‐transfer rate constant for laccase immobilized on carbon electrode is 3.4 s^?1. The bioelectrocatalytic activity of the enzyme was studied in the presence of 1,4‐hydroquinone (HQ). The kinetics of HQ oxidation is very fast (K_M=3.8 μM). However, the catalytic activity of laccase in the presence of high concentration of HQ decreases drastically. It is suggested that the T2/T3 copper center is able to accept electrons from HQ molecules directly via intramolecular channel.     

Mechanism of nitrite reduction at T2Cu centers: electronic structure calculations of catalysis by copper nitrite reductase and by synthetic model compounds

The mechanism of nitrite reduction at the Cu(II) center of both copper nitrite reductase and a number of corresponding synthetic models has been investigated by using both QM/MM and cluster calculations employing density functional theory methods. The mechanism in both cases is found to be very similar. Initially nitrite is bound in a bidentate fashion to the Cu(II) center via the two oxygen atoms. Upon reduction of the copper center, the two possible coordination modes of the protonated nitrite, by either nitrogen or a single oxygen atom, are close in energy, with nitrogen coordination probably preferred. Further protonation of this species leads to N-O bond cleavage, and an electron transfer from the Cu(I) center to the N-O+ ligand, resulting in loss of NO and regeneration of the resting state of the enzyme having a bound water molecule.     

A spectroscopic investigation of the interaction between nitrogen monoxide and copper sites of the fungal laccase from Rigidoporus lignosus

The interaction of NO, with the copper centres of the laccase secreted by Rigidoporus lignosus was studied under both aerobic or anaerobic conditions. The reduction of the T1 site was always observed, as detected by the disappearance of the characteristic optical band at 604 nm (T3 presents probably the same behaviour because of the decreasing of the band at 330 nm) and the absence of its characteristic EPR signal, while T2 undergoes an initial partial and transitory reduction, its EPR signal intensity totally restoring after 24 h interaction. Different magnetic parameters of the T2 site have been detected, evidencing an increase of the hyperfine coupling constant. Furthermore, the number of superhyperfine lines on the fourth line of T2 copper was also found to increase from seven in the native to nine in the NO-treated laccase, this fact implying the coordination of a nitrogenous species to the T2 site. It was also shown that nitrite can be a source of NO, thus, paralleling the behaviour of NO-donor molecules or NO gas, but after longer interaction times. The nitrogenous species coordinated to T2 site is probably NO2-, which arises indirectly by NO oxidation. In order to understand the mechanistic pathway of this interaction, some experiments were also carried out in the presence of azide to study the interaction of NO with this laccase having its trinuclear cluster blocked by the presence of an exogenous ligand as N3-. After the addition of NO-donor molecules to the azide-treated laccase, a new EPR signal appeared at low temperatures, which is ascribable to the partially reduced T3 site, while the T1 and T2 sites were found to be totally reduced. The mechanistic pathway of the NO interaction seems to proceed through the reduction of T1 and T3 copper sites, followed by the coordination of nitrogenous species to T2.     

The Resting Oxidized State of Small Laccase Analyzed with Paramagnetic NMR Spectroscopy

The enzyme laccase catalyzes the reduction of dioxygen to water at the trinuclear copper center (TNC). The TNC comprises a type-3 (T3) and a type-2 (T2) copper site. The paramagnetic NMR spectrum of the small laccase from Streptomyces coelicolor (SLAC) without the substrate shows a mixture of two catalytic states, the resting oxidized (RO) state and the native intermediate (NI) state. An analysis of the resonances of the RO state is reported. In this state, hydrogen resonances only of the T3 copper ligands can be found, in the region of 12–22 ppm. Signals from all six histidine ligands are found and can be attributed to Hδ1, Hβ or backbone amide H^N nuclei. Two sequence-specific assignments are proposed on the basis of a second-coordination shell variant that also lacks the copper ion at the T1 site, SLAC−T1D/Q291E. This double mutant is found to be exclusively in the RO state, revealing a subtle balance between the RO and the NI states.     

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.     

Role of ligand to control the mechanism of nitric oxide reduction of copper(II) complexes and ligand nitrosation

The nitric oxide reactivity of two copper(II) complexes, 1 and 2 with ligands L(1) and L(2), respectively, [L(1) = 5,5,7,12,12,14-hexamethyl-1,4,8,11-tetraazacyclotetradecane, L(2) = 5,5,7-trimethyl-[1,4]-diazepane] have been studied. The copper(II) center in complex 1 was found to be unreactive toward nitric oxide in pure acetonitrile; however, it displayed reduction in methanol solvent in presence of base. The copper(II) center in 2, in acetonitrile solvent, on exposure to nitric oxide has been found to be reduced to copper(I). The same reduction was observed in methanol, also, in case of complex 2. In case of complex 1, presumably, the attack of nitric oxide on the deprotonated amine is the first step, followed by electron transfer to the copper(II) center to afford the reduction. Alternatively, first NO coordination to the Cu(II) followed by NO(+) migration to the secondary amine is the most probable in case of complex 2. The observation of the transient intermediate in UV-visible and FT-IR spectroscopy prior to reduction in case of complex 2 also supports this possibility. In both cases, the reduction resulted into N-nitrosation; in 1, only mononitrosation was observed whereas complex 2 afforded dinitrosation as major product along with a minor amount of mononitrosation. Thus, it is evident from the present study that the macrocyclic ligands prefer the deprotonation pathway leading to mononitrosation; whereas nonmacrocyclic ones prefer the [Cu(II)-NO] intermediate pathway resulting into nitrosation at all the available sites of the ligand as major product.     

Dioxygen reduction by multi-copper oxidases; a structural perspective

The multi-copper oxidases oxidise substrate molecules by accepting electrons at a mononuclear copper centre and transferring them to a trinuclear centre. Dioxygen binds to the trinuclear centre and, following the transfer of four electrons, is reduced to two molecules of water. The precise mechanism of this reduction has been unclear, but recent X-ray structural studies using the CotA endospore coat protein from Bacillus subtilis have given further insights into the principal stages. It is proposed that the mechanism involves binding of the dioxygen into the trinuclear centre so that it is sited approximately symmetrically between the two type 3 copper ions with one oxygen atom close to the type 2 copper ion. Further stages involve the formation of a peroxide intermediate and following the splitting of this intermediate, the migration of the hydroxide moieties towards the solvent exit channel. The migration steps are likely to involve a movement of the type 2 copper ion and its environment. Details of a putative mechanism are described herein based both on structures already reported in the literature and on structures of the CotA protein in the oxidised and reduced states and with the addition of peroxide and the inhibitor, azide.     

Catalysis of novel enzymatic iodide oxidation by fungal laccase

A fungal laccase (Myceliophthom thermophila) has been shown to function as an iodide oxidase. Unlike other halides which interact with the type 2 copper site and are inhibitors for the laccase, iodide interacts with the type 1 copper site and serves as a substrate capable of donating an electron to the laccase. Under anaerobic conditions, the interaction between the laccase and iodide results in the reduction of the laccase type 1 copper and the concomitant oxidation of iodide to form iodide. In aerated solutions, the laccase catalyzes the oxidation of iodide to iodine and the concomitant reduction of dioxygen to water. The reaction exhibits typical Michaelis kinetics with aK _m of 0.16 ± 0.02M and ak _cat of 2.7 ± 0.2 turnovers per min at the optimal pH (3.4). The catalysis can be enhanced by 2,2′-azino-bis-(3-ethylbenz-thiazoline-6-sulfonic acid), which shuttles electrons rapidly between iodide and the laccase. Bilirubin oxidase also demonstrates significant iodide oxidase activity, suggesting that the property could be a common feature for copper-containing oxidases. Possible industrial and medicinal applications for a laccase-based iodine production system are discussed.     

Structural and functional mimic of galactose oxidase by a copper complex of a sterically demanding [N2O2] ligand

A structural and functional mimic of the galactose oxidase (GOase) enzyme active-site by a copper complex supported over a sterically demanding ligand having [N2O2] donor sites is reported. Specifically, the binding of the histidine (496 and 581) and tyrosine (272 and 495) residues to the copper center in a square-pyramidal fashion in the active-site of galactose oxidase (GOase) enzyme has been modeled in a copper complex, ([(3-tert-butyl-5-methyl-2-hydoxybenzyl)(3'-tert-butyl-5'-methyl-2'-oxobenzyl)(2-pyridylmethyl)]amine)Cu(OAc)) (1b), stabilized over a sterically demanding ligand in which the two phenolate-O atoms mimicked the tyrosine binding while an amine-N and pyridyl-N atoms emulated the histidine binding to the metal center, similar to that in the enzyme active-site. Furthermore, the copper complex 1b is found to be an effective functional model of the enzyme as it efficiently catalyzed the chemoselective oxidation of primary alcohols to aldehydes in high turnover numbers under ambient conditions. An insight into the nature of the active-species was obtained by EPR and CV studies, which in conjunction with the DFT studies, revealed that the active-species is an anti-ferromagnetically coupled diamagnetic radical cation, (1)1b+, obtained by one electron oxidation at the equatorial phenolate-O atom of the ligand in the 1b complex.     

Basic and applied features of multicopper oxidases, CueO, bilirubin oxidase, and laccase

Multicopper oxidases (MCOs) such as CueO, bilirubin oxidase, and laccase contain four Cu centers, type 1 Cu, type II Cu, and a pair of type III Cu's in a protein molecule consisting of three domains with homologous structure to cupredoxin containing only type I Cu. Type I Cu mediates electron transfer between the substrate and the trinuclear Cu center formed by a type II Cu and a pair of type III Cu's, where the final electron acceptor O(2) is converted to H(2)O without releasing activated oxygen species. During the process, O(2) is reduced by MCOs such as lacquer laccase and bilirubin oxidase; the reaction intermediate II with a possible doubly OH(-)-bridged structure in the trinuclear Cu center has been detected. The preceding reaction intermediate I has been detected by the reaction of the lacquer laccase in a mixed valence state, at which type I Cu was cuprous and the trinuclear Cu center was fully reduced, and by the reaction of the Cys --> Ser mutant for the type I Cu site in bilirubin oxidase and CueO. An acidic amino acid residue located adjacent to the trinuclear Cu center was proved to function as a proton donor to these reaction intermediates. The substrate specificity of MCO for organic substrates is produced by the integrated effects of the shape of the substrate-binding site and the specific interaction of the substrate with the amino acid located adjacent to the His residue coordinating to the type I Cu. In contrast, the substrate specificity of the cuprous oxidase, CueO, is produced by the segment covering the Cu(I)-binding site so as to obstruct the access of organic substrates. Truncating the segment spanning helix 5 to helix 7 greatly reduced the specificity of CueO for Cu(I) and prominently enhanced the low oxidizing activity for the organic substrates, indicating the success of protein engineering to modify the substrate specificity of MCO.     

Properties of Pd---Au and Pt---Cu alloy surfaces for the adsorption and catalytic reduction of O2 and NO by H2

The adsorption and catalytic properties of Pd---Au and Pt---C alloy surfaces were investigated under low pressure conditions, with the real surface composition being monitored using Auger electron spectroscopy. Flash desorption experiments on O₂ and NO, and steady state kinetic experiments involving the reduction of these substances by H₂ were performed on polycrystalline alloy surfaces. For the Pd---Au system, O₂ desorption was promoted by gold, as shown by the shift of O₂ desorption toward lower temperature and a linear decrease in the saturated amount of O₂ adsorption with increase in gold content. For the Pt---Cu system, O₂ desorption was retarded by copper i.e. the O₂ desorption temperature shifted upward with increase in copper content. In both the alloy systems, catalytic activities for the above reactions were significantly suppressed by the addition of gold or copper to the platinum group metals. The drop was more pronounced for NO reduction, suggesting that a larger ensemble of active platinum group metal atoms are necessary for NO dissociation.     

Determinants of the relative reduction potentials of type-1 copper sites in proteins

The relative Cu(2+)/Cu(+) reduction potentials of six type-1 copper sites (cucumber stellacyanin, P. aeruginosa azurin, poplar plastocyanin, C. cinereus laccase, T. ferrooxidans rusticyanin, and human ceruloplasmin), which lie in a reduction potential range from 260 mV to over 1000 mV, have been studied by quantum mechanical calculations. The range and relative orderings of the reduction potentials are reproduced very well compared to experimental values. The study suggests that the main structural determinants of the relative reduction potentials of the blue copper sites are located within 6 A of the Cu atoms. Further analysis suggests that the reduction potential differences of type-1 copper sites are caused by axial ligand interactions, hydrogen bonding to the S(Cys), and protein constraint on the inner sphere ligand orientations. The low reduction potential of cucumber stellacyanin is due mainly to a glutamine ligand at the axial position, rather than a methionine or a hydrophobic residue as in the other proteins. A stronger interaction with a backbone carbonyl group is a prime contributor to the lower reduction potential of P. aeruginosa azurin as compared to poplar plastocyanin, whereas the reverse is true for C. cinereus laccase and T. ferrooxidans rusticyanin. The lack of an axial methonine ligand also contributes significantly to the increased reduction potentials of C. cinereus laccase and human ceruloplasmin. However, in the case of C. cinereus laccase, this increase is attenuated by the presence of only one amide NH hydrogen bond to the S(Cys) rather than two in the other proteins. In human ceruloplasmin the reduction potential is further increased by the structural distortion of the equatorial ligand orientation.     

Application of copper(I) halides to modifying reactivity of polyhalomethanes and arenesulfonyl chlorides in free-radical addition. “Cross-halogenation” reaction

In the free-radical addition of a number of organohalogen reagents to cyclic alkenes and dienes in the presence of copper(I) halides the composition of the reaction products is governed by the stage of a fast halogen transfer from the copper derivative to the alkyl radical. Under these conditions in contrast to the free-radical addition reactions initiated by UV light or peroxide initiators the intramolecular rearrangements are suppressed, the stereoselectivity of the reaction changes, and also some adducts contain halogen atoms different from those present in the organohalogen reagent employed.     

Reaction of a copper(II)-nitrosyl complex with hydrogen peroxide: putative formation of a copper(I)-peroxynitrite intermediate

The reaction of a Cu(II)-nitrosyl complex (1) with hydrogen peroxide at -20 °C in acetonitrile results in the formation of the corresponding Cu(I)-peroxynitrite intermediate. The reduction of the Cu(II) center was monitored by UV-visible spectroscopic studies. Formation of the peroxynitrite intermediate has been confirmed by its characteristic phenol ring nitration reaction as well as isolation of corresponding Cu(I)-nitrate (2). On air oxidation, 2 resulted in the corresponding Cu(II)-nitrate (3). Thus, these results demonstrate a possible decomposition pathway for H(2)O(2) and NO through the formation of a peroxynitrite intermediate in biological systems.     

Intersite structural rearrangement of the blue copper site induced by substrate binding: spectroscopic studies of a copper-containing nitrite reductase from Alcaligenes xylosoxidans NCIMB 11015

A copper-containing nitrite reductase from Alcaligenes xylosoxidans NCIMB 11015 has its own unique blue or type 1 copper protein resonance Raman spectrum in the usual Cu-S(Cys) stretching region, nu(Cu-S(Cys)), with a pair of strong peaks at 412 and 420 cm(-1) and a weak peak at 364 cm(-1). The predominantly nu(Cu-S(Cys)) Raman bands at 412, 420, and 364 cm(-1) of the type 1 copper site all shifted to higher frequencies upon binding of nitrite to the type 2 copper site, and the resonance Raman difference spectra progressively intensified with the increments of nitrite ion concentration. Positive support for substrate binding to the type 2 copper is provided by the nu(Cu-S(Cys)) bands in the resonance Raman spectrum of a type 2 copper-depleted enzyme, which is insensitive to the presence of NO2-. The shift to higher frequency of the Raman bands of the type 1 copper center with the addition of nitrite ions suggests a stronger Cu-S(Cys) interaction in the substrate-bound A. xylosoxidans nitrite reductase.     

Copper(I) Nitro Complex with an Anionic [HB(3,5-Me(2)Pz)(3)](-) Ligand: A Synthetic Model for the Copper Nitrite Reductase Active Site

The new copper(I) nitro complex [(Ph(3)P)(2)N][Cu(HB(3,5-Me(2)Pz)(3))(NO(2))] (2), containing the anionic hydrotris(3,5-dimethylpyrazolyl)borate ligand, was synthesized, and its structural features were probed using X-ray crystallography. Complex 2 was found to cocrystallize with a water molecule, and X-ray crystallographic analysis showed that the resulting molecule had the structure [(Ph(3)P)(2)N][Cu(HB(3,5-Me(2)Pz)(3))(NO(2))]·H(2)O (3), containing a water hydrogen bonded to an oxygen of the nitrite moiety. This complex represents the first example in the solid state of an analogue of the nitrous acid intermediate (CuNO(2)H). A comparison of the nitrite reduction reactivity of the electron-rich ligand containing the CuNO(2) complex 2 with that of the known neutral ligand containing the CuNO(2) complex [Cu(HC(3,5-Me(2)Pz)(3))(NO(2))] (1) shows that reactivity is significantly influenced by the electron density around the copper and nitrite centers. The detailed mechanisms of nitrite reduction reactions of 1 and 2 with acetic acid were explored by using density functional theory calculations. Overall, the results of this effort show that synthetic models, based on neutral HC(3,5-Me(2)Pz)(3) and anionic [HB(3,5-Me(2)Pz)(3)](-) ligands, mimic the electronic influence of (His)(3) ligands in the environment of the type II copper center of copper nitrite reductases (Cu-NIRs).     

A New <Emphasis Type="Italic">Stenotrophomonas maltophilia</Emphasis> Strain Producing Laccase. Use in Decolorization of Synthetics Dyes

Laccase activity was detected in a soil bacterium Stenotrophomonas maltophilia AAP56 identified by biochemical and molecular methods. It was produced in cells at the stationary growth phase in Luria Bertani (LB) medium added by 0.4 mM copper sulfate. The addition of CuSO₄ in culture medium improved production of laccase activity. However, one laccase enzyme was detected by native polyacrylamide gel electrophoresis. The enzyme showed syringaldazine (K _m = 53 μM), 2,2’-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (K _m = 700 μM), and pyrocatechol (K _m = 25 μM) oxidase activity and was activated by addition of 0.1% (v/v) Triton-X-100 in the reaction mixture. Moreover, the laccase activity was increased 2.6-fold by the addition of 10 mM copper sulfate; the enzyme was totally inhibited by ethylenediaminetetraacetic acid (5 mM), suggesting that this laccase is a metal-dependant one. Decolorization activity of some synthetic dyes (methylene blue, methyl green, toluidine blue, Congo red, methyl orange, and pink) and the industrial effluent (SITEX Black) was achieved by the bacteria S. maltophilia AAP56 in the LB growth medium under shaking conditions.     

Nitrogen oxide interaction with copper complexes formed by small peptides belonging to the prion protein octa-repeat region

The interaction between NO and copper(II) complexes formed by peptides coming from the N-terminal prion protein octa-repeat region was studied. Aqueous solutions of the Cu-Ac-HGGG-NH(2) and the Cu-Ac-PHGGGWGQ-NH(2) systems around pH 7.5 were tested after the addition of NONOates as a source of NO. UV-Vis, room temperature and frozen solution EPR spectra showed the occurrence of copper(ii) reduction in all these complexes. The reduction of these complexes is probably mediated by the formation of a labile NO adduct, which, after re-oxidation, leads to a relatively stable NO(2)(-) adduct through the apical coordination along the void site of their square pyramidal structure. In fact, the most significant shifts in EPR magnetic parameters (g(||) and A(||) or g(iso) and A(iso)) as well as in the optical parameters (lambda(max) and epsilon(max)) gave a reason for geometrical changes of the copper coordination polyhedron from a distorted square pyramid to a pseudo-octahedron. The presence of oxygen in the aqueous solution hindered the reduction ability of NO towards copper, but it made it easier to return to the original species. In order to elucidate the possible mechanism of this interaction, the reduction of copper complexed by these ligands was followed by means of zinc powder addition. The further addition of nitrite to the solution containing reduced copper led to the conclusion that nitrite could easily form an adduct, which after re-oxidation presented the same spectral features of the species obtained when the NO interaction was followed. The complexity of this interaction could involve both an inner or an outer-sphere electron transfer mechanism.