Trinuclear Copper Cluster Research Articles

Ligand regulation and capacitive properties of nickel based pyrazole trinuclear copper cluster organic framework

Traditional metal organic framework (MOF) hinders its application as high-performance electrode materials for supercapacitors due to the limited electron transfer efficiency and conjugation of its ligand molecules. Therefore, in this article, highly electrochemical active pyrazole trincore-copper cluster derivatives as the core unit were covalently assembled by Schiff base reaction with organic linkers of different structures to obtain a series of structurally different electroactive ligands. It was found that ligands with multiactive metal chelation site structure with polydentate carboxylate linker and aromatic extension showed high coordination ability and high degree of conjugation. The nickel-based MOF synthesized with this novel electroactive ligand exhibited a high specific capacitance of 1804 Fg−1 at a current density of 1 Ag−1. At a power density of 747.25 W kg−1, the maximum energy density of the assembled asymmetric supercapacitor assembled with Cu-PyAc-Am2Ac-Ni is 58.95 Wh kg−1 with cycling stability of 88.95 % after 5000 charges and discharges. This means that Cu-PyAc-Am2Ac-Ni is a promising electrode material for supercapacitors.

Trinuclear Cu-based covalent organic framework: π-conjugated framework regulating electron delocalization to promote photoreduction CO2

Sunlight-driven CO2 reduction to value-added chemicals is an effective strategy to promote carbon recycling. The exploration of catalysts with efficient charge separation is crucially important for highly efficient CO2 photoreduction. In this work, the preparation of metal-cluster-based covalent organic framework (CuABD) integrated features from both metal organic frameworks (MOFs) and covalent organic frameworks (COFs) through the condensation of diamines and functionalized trinuclear copper clusters demonstrate a thoughtful design strategy. The reported yield of 1.3 mmol g−1 h−1 for formic acid (HCOOH) under simulated solar irradiation is impressive, surpassing the performance of many COF- and MOF-based catalysts previously reported. Compared to its isomorphic metal-free structure (named BDFTD) and bare trinuclear Cu cluster which present extremely poor catalytic activities, CuABD displays remarkably enhanced CO2 reduction activity. Experimental and theoretical investigations reveal that the efficient charge transfer between diamine monomer and cyclic trinuclear copper (I) units, and the electron delocalization of the π-conjugated framework are responsible for the appealing catalytic performance. In summary, the work presents a well-structured and scientifically sound exploration of a metal-cluster-based covalent organic framework for efficient CO2 reduction under sunlight.

Engineering Copper‐Based Covalent Organic Framework Microenvironments to Enable Efficient CO2 Electroreduction with Tunable Ethylene/Methane Switch

AbstractA microenvironment engineering strategy has been developed to switch the CO2 electroreduction reaction (CO2RR) selectivity from methane (CH4) to ethylene (C2H4) by adjusting the coordination microstructures of trinuclear copper cluster‐based metal‐covalent organic framework (MCOF). When Cu sites are oriented to channels in Cu‐PyCA‐MCOF, methane is the main product. Conversely, when trinuclear copper sites are coordinated with OH− and H2O molecules in Cu‐PyCAOH‐MCOF nanosheets, the main product switches from CH4 to C2H4 with 50.5% selectivity and 200.2 mA cm− partial current density at −1.2 V (vs RHE). This happens because CO2 molecules can only contact active sites perpendicular to the trinuclear copper cluster plane in Cu‐PyCAOH‐MCOF nanosheets, where the Cu─Cu distance between them is 3.2 Å, favoring the efficient conversion of CO2 to C2H4 through the C─C coupling reaction. Operando infrared spectroscopy, in situ X‐ray absorption near‐edge structure spectra, and DFT calculations reveal that changing the coordination environments of MCOFs significantly stabilizes key intermediates and reduces the energies of the CO2RR. This work offers an effective strategy for enhancing CO2RR performance toward C2H4 products by tuning the microenvironments of copper‐based electrocatalysts.

Switching the Symmetry of a Trinuclear Copper Cluster Catalyst for Electroreducing CO2 to an Asymmetric C2 Product in an Acidic Electrolyte

Switching the Symmetry of a Trinuclear Copper Cluster Catalyst for Electroreducing CO₂ to an Asymmetric C₂ Product in an Acidic Electrolyte

Hydrophobic Trinuclear Copper Cluster-Containing Organic Framework for Synergetic Electrocatalytic Synthesis of Amino Acids.

Electrocatalytic synthesis of amino acids provides a promising green and efficient pathway to manufacture the basic substances of life. Herein, reaction of 2,5-perfluroalkyl-terepthalohydrazide and tris(4-µ2 -O-carboxaldehyde-pyrazolato-N, N')-tricopper affords a crystalline trinuclear copper cluster-containing organic framework, named F-Cu3 -OF. Incorporation of abundant hydrophobic perfluroalkyl groups inside the channels of F-Cu3 -OF is revealed to successfully suppress the hydrogen evolution reaction via preventing H+ cation with large polarity from the framework of F-Cu3 -OF and in turn increasing the adsorption of other substrates with relatively small polarity like NO3 - and keto acids on the active sites. The copper atoms with short distance in the trinuclear copper clusters of F-Cu3 -OF enable simultaneous activization of NO3 - and keto acids, facilitating the following synergistic and efficient C─N coupling on the basis of in situ spectroscopic investigations together with theoretical calculation. Combination of these effects leads to efficient electroproduction of various amino acids including glycine, alanine, leucine, valine, and phenylalanine from NO3 - and keto acids with a Faraday efficiency of 42%-71% and a yield of 187-957µmol cm-2 h-1 , representing the thus far best performance. This work shall be helpful for developing economical, eco-friendly, and high-efficiency strategy for the production of amino acids and other life substances.

Structural Insight into the Amino Acid Environment of the Two-Domain Laccase's Trinuclear Copper Cluster.

Laccases are industrially relevant enzymes. However, their range of applications is limited by their functioning and stability. Most of the currently known laccases function in acidic conditions at temperatures below 60 °C, but two-domain laccases (2D) oxidize some substrates in alkaline conditions and above 70 °C. In this study, we aim to establish the structural factors affecting the alkaline activity of the 2D laccase from Streptomyces griseoflavus (SgfSL). The range of methods used allowed us to show that the alkaline activity of SgfSL is influenced by the polar residues located close to the trinuclear center (TNC). Structural and functional studies of the SgfSL mutants Met199Ala/Asp268Asn and Met199Gly/Asp268Asn revealed that the substitution Asp268Asn (11 Å from the TNC) affects the orientation of the Asn261 (the second coordination sphere of the TNC), resulting in hydrogen-bond-network reorganization, which leads to a change in the SgfSL-activity pH profile. The combination of the Met199Gly/Arg240His and Asp268Asn substitutions increased the efficiency (kcat/KM) of the 2,6-DMP oxidation by 34-fold compared with the SgfSL. Our results extend the knowledge about the structure and functioning of 2D laccases' TNC active sites and open up new possibilities for the directed engineering of laccases.

Mechanistic Investigations into the Selective Reduction of Oxygen by a Multicopper Oxidase T3 Site-Inspired Dicopper Complex.

Understanding how multicopper oxidases (MCOs) reduce oxygen in the trinuclear copper cluster (TNC) is of great importance for development of catalysts for the oxygen reduction reaction (ORR). Herein, we report a mechanistic investigation into the ORR activity of the dinuclear copper complex [Cu2L(μ-OH)]3+ (L = 2,7-bis[bis(2-pyridylmethyl)aminomethyl]-1,8-naphthyridine). This complex is inspired by the dinuclear T3 site found in the MCO active site and confines the Cu centers in a rigid scaffold. We show that the electrochemical reduction of [Cu2L(μ-OH)]3+ follows a proton-coupled electron transfer pathway and requires a larger overpotential due to the presence of the Cu-OH-Cu motif. In addition, we provide evidence that metal-metal cooperativity takes place during catalysis that is facilitated by the constraints of the rigid ligand framework, by identification of key intermediates along the catalytic cycle of [Cu2L(μ-OH)]3+ . Electrochemical studies show that the mechanisms of the ORR and hydrogen peroxide reduction reaction found for [Cu2L(μ-OH)]3+ differ from the ones found for analogous mononuclear copper catalysts. In addition, the metal-metal cooperativity results in an improved selectivity for the four-electron ORR of more than 70% because reaction intermediates can be stabilized better between both copper centers. Overall, the mechanism of the [Cu2L(μ-OH)]3+ -catalyzed ORR in this work contributes to the understanding of how the cooperative function of multiple metals in close proximity can affect ORR activity and selectivity.

The Boundary between Two Modes of Gas Evolution: Oscillatory (H2 and O2) and Conventional Redox (O2 Only), in the Hydrocarbon/H2O2/Cu(II)/CH3CN System

During the oxidation of hydrocarbons using hydrogen peroxide solutions, the evolution of gaseous oxygen is a side and undesirable process, in which the consumption of the oxidizer is not associated with the formation of target products. Therefore, no attention is paid to the systematic study of the chemical composition of the gas and the mechanisms of its formation. Filling this gap, the authors discovered a number of new, previously unidentified, interesting facts concerning both gas evolution and the oxidation of hydrocarbons. In a 33% H2O2/Cu2Cl4·2DMG/CH3CN system, where DMG is dimethylglyoxime (Butane-2,3-dione dioxime), and is at 50 °C, evidence of significant evolution of gaseous hydrogen, along with the evolution of gaseous oxygen was found. In the authors’ opinion, which requires additional verification, the ratio of gaseous hydrogen and oxygen in the discussed catalytic system can reach up to 1:1. The conditions in which only gaseous oxygen is formed are selected. Using a number of oxidizable hydrocarbons with the first adiabatic ionization potentials (AIPs) of a wide range of values, it was found that the first stage of such a process of evolving only gaseous oxygen was the single electron transfer from hydrogen peroxide molecules to trinuclear copper clusters with the formation, respectively, of hydrogen peroxide radical cations H2O2•+ and radical anions Cu3Cl5•− (AIP = 5 eV). When the conditions for the implementation of such a single electron transfer mechanism are exhausted, the channel of decomposition of hydrogen peroxide molecules into gaseous hydrogen and oxygen is switched on, which is accompanied by the transition of the system to an oscillatory mode of gas evolution. In some cases, the formation of additional amounts of gaseous products is provided by the catalytically activated decomposition of water molecules into hydrogen and oxygen after the complete consumption of hydrogen peroxide molecules in the reaction of gaseous oxygen evolution. The adiabatic electron affinity of various forms of copper molecules involved in chemical processes is calculated by the density functional theory method.

Multiple Proton-Coupled Electron Transfers at a Tricopper Cluster: Modeling the Reductive Regeneration Process in Multicopper Oxidases.

Metal clusters in enzymes carry out the life-sustaining reactions by accumulating multiple redox equivalents in a narrow potential range. This redox potential leveling effect commonly observed in Nature has yet to be reproduced with synthetic metal clusters. Herein, we employ a fully encapsulated synthetic tricopper complex to model the three-electron two-proton reductive regeneration of fully reduced trinuclear copper cluster CuICuICuI(μ2-OH2) (FR) from native intermediate CuIICuIICuII(μ3-O) (NI) in multicopper oxidases (MCOs). The tricopper cluster can access four oxidation states (I,I,I to II,II,II) and four protonation states ([Cu3(μ3-O)]LH, [Cu3(μ3-OH)]L, [Cu3(μ3-OH)]LH, and [Cu3(μ3-OH2)]L, where LH denotes the protonated ligand), allowing mechanistic investigation of proton-coupled electron transfer (PCET) relevant to MCOs. Seven tricopper complexes with discrete oxidation and protonation states were characterized with spectroscopy or X-ray single-crystal diffraction. A stepwise electron transfer-proton transfer (ET-PT) mechanism is established for the reduction of CuIICuIICuII(μ3-O)LH to CuIICuIICuI(μ3-OH)L, while a stepwise PT-ET mechanism is determined for the reduction of CuIICuICuI(μ3-OH)LH to CuICuICuI(μ2-OH2)L. The switch-over from ET-PT to PT-ET mechanism showcases that the tricopper complex can adopt different PCET mechanisms to circumvent high-barrier proton transfer steps. Overall, three-electron two-proton reduction occurs within a narrow potential range of 170 mV, exemplifying the redox potential leveling effect of secondary proton relays in delivering multiple redox equivalents at metal clusters.

Bioinspired Trinuclear Copper Catalyst for Water Oxidation with a Turnover Frequency up to 20000 s-1.

Solar-powered water splitting is a dream reaction for constructing an artificial photosynthetic system for producing solar fuels. Natural photosystem II is a prototype template for research on artificial solar energy conversion by oxidizing water into molecular oxygen and supplying four electrons for fuel production. Although a range of synthetic molecular water oxidation catalysts have been developed, the understanding of O-O bond formation in this multielectron and multiproton catalytic process is limited, and thus water oxidation is still a big challenge. Herein, we report a trinuclear copper cluster that displays outstanding reactivity toward catalytic water oxidation inspired by multicopper oxidases (MCOs), which provides efficient catalytic four-electron reduction of O2 to water. This synthetic mimic exhibits a turnover frequency of 20000 s-1 in sodium bicarbonate solution, which is about 150 and 15 times higher than that of the mononuclear Cu catalyst (F-N2O2Cu, 131.6 s-1) and binuclear Cu2 complex (HappCu2, 1375 s-1), respectively. This work shows that the cooperation between multiple metals is an effective strategy to regulate the formation of O-O bond in water oxidation catalysis.

Predesign of Catalytically Active Sites via Stable Coordination Cluster Model System for Electroreduction of CO2 to Ethylene.

Purposefully designing the well-defined catalysts for the selective electroreduction of CO2 to C2 H4 is an extremely important but challenging work. In this work, three crystalline trinuclear copper clusters (Cu3 -X, X=Cl- , Br- , NO3 - ) have been designed, containing three active Cu sites with the identical coordination environment and appropriate spatial distance, delivering high selectivity for the electrocatalytic reduction of CO2 to C2 H4 . The highest faradaic efficiency of Cu3 -X for CO2 -to-C2 H4 conversion can be adjusted from 31.90 % to 55.01 % by simply replacing the counter anions (NO3 - , Cl- , Br- ). The DFT calculation results verify that Cu3 -X can facilitate the C-C coupling of identical *CHO intermediates, subsequently forming molecular symmetrical C2 H4 product. This work provides an important molecular model system and a new design perspective for electroreduction of CO2 to C2 products with symmetrical molecular structure.

Predesign of Catalytically Active Sites via Stable Coordination Cluster Model System for Electroreduction of CO2 to Ethylene

AbstractPurposefully designing the well‐defined catalysts for the selective electroreduction of CO2 to C2H4 is an extremely important but challenging work. In this work, three crystalline trinuclear copper clusters (Cu3‐X, X=Cl−, Br−, NO3−) have been designed, containing three active Cu sites with the identical coordination environment and appropriate spatial distance, delivering high selectivity for the electrocatalytic reduction of CO2 to C2H4. The highest faradaic efficiency of Cu3‐X for CO2‐to‐C2H4 conversion can be adjusted from 31.90 % to 55.01 % by simply replacing the counter anions (NO3−, Cl−, Br−). The DFT calculation results verify that Cu3‐X can facilitate the C−C coupling of identical *CHO intermediates, subsequently forming molecular symmetrical C2H4 product. This work provides an important molecular model system and a new design perspective for electroreduction of CO2 to C2 products with symmetrical molecular structure.

Electron Transfer to the Trinuclear Copper Cluster in Electrocatalysis by the Multicopper Oxidases.

High-potential multicopper oxidases (MCOs) are excellent catalysts able to perform the oxygen reduction reaction (ORR) at remarkably low overpotentials. Moreover, MCOs are able to interact directly with the electrode surfaces via direct electron transfer (DET), that makes them the most commonly used electrocatalysts for oxygen reduction in biofuel cells. The central question in MCO electrocatalysis is whether the type 1 (T1) Cu is the primary electron acceptor site from the electrode, or whether electrons can be transferred directly to the trinuclear copper cluster (TNC), bypassing the rate-limiting intramolecular electron transfer step from the T1 site. Here, using site-directed mutagenesis and electrochemical methods combined with data modeling of electrode kinetics, we have found that there is no preferential superexchange pathway for DET to the T1 site. However, due to the high reorganization energy of the fully oxidized TNC, electron transfer from the electrode to the TNC does occur primarily through the T1 site. We have further demonstrated that the lower reorganization energy of the TNC in its two-electron reduced, alternative resting, form enables DET to the TNC, but this only occurs in the first turnover. This study provides insight into the factors that control the kinetics of electrocatalysis by the MCOs and a guide for the design of more efficient biocathodes for the ORR.

Role of a Tyrosine Radical in Human Ceruloplasmin Catalysis.

Multicopper oxidases (MCOs) are a large family of diverse enzymes found in both eukaryotes and prokaryotes that couple one-electron oxidations of various substrates to the four-electron reduction of O2 to H2O, functioning through a set of metallocofactors consisting of one type 1 copper (T1 Cu) and one trinuclear copper cluster (TNC). Human serum ceruloplasmin (Cp) is a unique member of MCOs composed of six cupredoxin domains and harbors six Cu ions arranged as three T1 Cu and one TNC. The native substrate of Cp is Fe2+. It is an essential ferroxidase critical for iron homeostasis and is closely associated with metal-mediated diseases and metal neurotoxicity. In human serum, Cp operates under substrate-limiting low [Fe2+] but high [O2] conditions, implying the possible involvement of partially reduced intermediates in Cp catalysis. In this work, we studied for the first time Cp reactivities at defined partially reduced states and discovered a tyrosine radical weakly magnetically coupled to the native intermediate (NI) of the TNC via a hydrogen bond. Our results lead to a new hypothesis that human iron transport is regulated as the paired transfer of iron from ferroportin to Cp to transferrin, and the tyrosine residue in Cp acts as a gate to avoid reactive oxygen species (ROS) formation when Fe2+ delivery is dysregulated.

Molecular dynamics analysis of conserved water mediated inter-domain recognition of His667-Trp669 in human ceruloplasmin.

The human ceruloplasmin (hCP) is the copper containing ferroxidase enzyme with multifunctional activities (NO-oxidase, NO2-synthase,oxidation of neurotransmitters including antioxidants). Therefore, it is of interest to probe the multi-domain hCP using moleculardynamics simulation. Results explain the role played by several conserved water centers in the intra and inter-domain recognition throughH-bond interaction with the interacting residues. We observed seventeen conserved water centers in the inter-domain recognition. Weshow that five invariant water centers W13, W14, W18, W23 and W26 connect the Domain 5 to Domain 4 (D5…W…W4). We also show thatfive other water centers W19, W20, W27, W30 and W31 connects the Domain 5 to Domain 6 (D5…W…W6) that is unique in the simulatedform. The W7 and W32 water centers are involved in the D1…W…W6 recognition. This is important for the water-mediated interaction ofGlu1032 to the trinuclear copper cluster present at the interface between these domains. The involvement of W10 water center in theD3…W10…D4 recognition through Gln552…W10…His667 H-bond interaction is critical in the complexation of CP with myeloperoxidase(Mpo). These observations provide insights to the molecular recognition of hCP with other biomolecules in the system.

Insights from molecular dynamics simulation of human ceruloplasmin (ferroxidase enzyme) binding with biogenic monoamines.

Human ceruloplasmin (hCP) is a multi-copper oxidase with ferroxidase and amine oxidase activities. Molecular dynamics simulation (MDS) and docking analysis of biogenic monoamines with ceruloplasmin explain the role of Asp1025, Glu935, Glu272, Glu232 and Glu230 together with the binding site water molecules (referred as conserved water molecules) in the stabilization of neurotransmitter (Serotonin, Norepinephrine and Epinephrine) molecules within the binding cavity of hCP. Conserved water molecules are found at specific positions interacting with the protein structures that have sequence similarity. The ethylamine side chain nitrogen atom (N1) of neurotransmitter molecules interacts with water molecules in the binding cavity formed by Asp1025, Glu935 and Glu232 residues. These residues form an acidic triad mimicking a substrate binding cavity. The hydroxyl groups attached to the catechol ring of epinephrine and norepinephrine have been stabilized by Asp230 and Asp232 residues. Data suggests that the recognition of biogenic amines mediates through the N+(amine) ...Asp1025-His1026-CuCis-His path. The potential recognition path of biogenic monoamines to trinuclear copper cluster supported by active site water molecules (referred as conserved water molecules) is described in this report.

O2 Reduction to Water by High Potential Multicopper Oxidases: Contributions of the T1 Copper Site Potential and the Local Environment of the Trinuclear Copper Cluster.

High potential multicopper oxidases (MCOs) have T1 reduction potentials >600 mV (vs normal hydrogen electrode), making them important catalysts for O2 reduction in various biotechnological applications. The oxygen reduction mechanism for the low potential MCOs is well-characterized; however, O2 reactivity of high potential MCOs is not well understood. In this study, we have shown that laccase from Trametes versicolor, where the T1 redox potential is increased by ∼350 mV over that of the low potential MCOs corresponding to an 8 kcal/mol decrease in the driving force, exhibits a slower intramolecular electron transfer (IET) rate to the trinuclear Cu cluster (TNC) in the native intermediate (NI), relative to the low potential MCO from Rhus vernicifera laccase. This IET rate is, however, >102 times faster than the decay rate of the NI, demonstrating that this intermediate form of the enzyme is catalytically relevant enabling fast turnover. However, in contrast to the low potential MCOs where T1 reduction by substrate is rate limiting, the rate limiting step in turnover of high potential MCOs is the first IET to NI. Part of the reduction potential difference of the T1 sites in high vs low potential MCOs is balanced by an ∼100 mV higher reduction potential of NI due to the more positive protein environment in the vicinity of the TNC.

A 2-D Metal–Organic Framework Based on Linear Trinuclear Copper Cluster Units: Syntheses, Structures and Magnetic Properties

The coordination polymer [Cu3(tci)2(phen)2] n (H3tci = 1,3,5-tris(2-carboxyethyl)isocyanurate; phen = 1,10-phenanthroline) has been synthesised. Single-crystal X-ray diffraction studies have shown that the polymer displays a 2-D metal–organic framework based on linear trinuclear copper cluster units, and its magnetic properties indicate antiferromagnetic interactions.

Four second-sphere residues of Thermus thermophilus SG0.5JP17-16 laccase tune the catalysis by hydrogen-bonding networks

The multicopper oxidases catalyze 1-electron oxidation of four substrate molecules and concomitantly 4-electron reduction of dioxygen to water. The substrate loses the electrons at the type 1 copper (T1 Cu) site of the enzyme, while the dioxygen is reduced to water at the trinuclear copper center. A highly conserved Glu residue, which is at the dioxygen-entering channel, shuttles the proton to break the O-O bond of dioxygen. At the water-leaving channel, an Asp residue was found to be important in the protonation mechanism. In this study, laccase from Thermus thermophilus SG0.5JP17-16 (lacTT) was investigated to address how four second-sphere residues E356, E456, D106, and D423 affect the activity of the enzyme. Kinetic data indicate that catalytic activities of the enzyme are altered by site-directed mutagenesis on four second-sphere residues. The structural model of lacTT was generated by homology modeling. Structural and spectral data indicate that the E356 residue is situated at the substrate-binding site, responsible for the binding of the substrate and the geometry of the T1 Cu site by hydrogen-bonding networks; the E456 residue, located at the dioxygen-entering channel, plays a critical role in stabilizing the structure of all active copper centers and shuttling the proton to the trinuclear copper cluster (TNC) for the reductive reaction of dioxygen; the D106 and D423 residues are at the water-leaving channel, and they are important for the essential geometry of the TNC and the release of the water molecules. Altogether, this study contributes to the further understanding of the basic mechanism involving the oxidation of the substrate, electron transfer, and the reduction of dioxygen in lacTT.

Recognition dynamics of trinuclear copper cluster and associated histidine residues through conserved or semi-conserved water molecules in human Ceruloplasmin: The involvement of aspartic and glutamic acid gates

Human Ceruloplasmin belongs to the family of multi-copper oxidases and it is involved in different physiological processes, copper ion transport, iron metabolism, iron homeostasis, and biogenic amine metabolism. MD-simulation studies have indicated the higher hydrophilic susceptibility of the trinuclear copper cluster in native CP compared to its oxygen bound form. The copper (T2/T3) atom Cu3047 of the cluster, which is close to T1 copper center Cu3052 (~13 Å) has a higher affinity for water molecules compared to other copper centers. The water molecules of W3, W4, W5, W9, and W12 conserved water sites are coordinated to Cu3047, where W3, W9, and W12 centers are found to play some crucial role in the stabilization of native trinuclear copper cluster. The hydrogen bonding interaction of Asp169, Glu112, Asp995, and Glu1032 residues with the copper-bound conserved water molecules (W3, W4, W5, W10, and W11) in native CP is observed to be unique. The conformational flexibility of Asp169 and Glu112 and their association with the copper-bound water molecules, but the absence of such interaction in O2-bound simulated structure of the enzyme is indicating some plausible rational on the role of these acidic residues in the gating of O2 molecule in the native trinuclear Cu cluster of CP. The simulation results may shade some new light on the biochemistry/chemistry of CP, specially on the hydration dynamics of the trinuclear copper cluster.