Binuclear Copper Center Research Articles

High pressure-derived nonsymmetrical [Cu2O]2+ core for room-temperature methane hydroxylation.

Nonsymmetrical oxygen-bridged binuclear copper centers have been proposed and modeled as intermediates and transition states in several C─H oxidation pathways, leading to the postulation that structural dissymmetry enhances the reactivity of the bridging oxygen. However, experimentally characterizing the structure and reactivity of these transient species is remarkably challenging. Here, we report the high-pressure synthesis of a metastable nonsymmetrical dicopper-μ-oxo compound with exceptional reactivity toward the mono-oxygenation of aliphatic C─H bonds. The nonequivalent coordination environment of copper stabilizes localized mixed valency and greatly enhances the hydrogen atom abstraction activity of the bridging oxygen, enabling room-temperature hydroxylation of methane under pressure. These findings highlight the role of dissymmetry in the reactivity of binuclear copper centers and demonstrate precise control of molecular structures by mechanical means.

Electro-enzymatic processes in mitochondria

Four enzymatic complexes form the electron transport chain of the mitochondrion. Complexes I and II are current generator half-elements, that create electron flows with the participation of the enzymes NADH dehydrogenase and Succinate dehydrogenase, oxidizing NADH·H into NAD+, and succinate into fumarate, respectively. Electrons are transferred throw the iron-sulfur clusters system, participate reduction enzymatic reaction of CoQ10 into CoQ10Н2. The proton pumping into the mitochondrial intermembrane space, as well as the regeneration of CoQ10 occur when electrons transferred by ubiquinone-cytochrome-c-oxidoreductase enzyme to the Riske iron-sulfur protein (Fe2S2). The Riske protein regulates electron flows: it transfers one electron to the bL,bН (III) complex, and the other to cytochrome c. Cytochrome c directs electrons to the ΙV complex electrolysis system. The binuclear copper center [CuA-CuB] of the ΙV complex is the anode, where oxygen is formed. Electrons along the galvanic pair chain from paired copper Cu2++ e- ↔ Cu+ and iron Fe2+- е- ↔Fe3+ are transferred to the cathode, copper ion (Сu+), where the electrochemical reaction occurs O2 + 4e- + 4H+ → 2H2O, with the pumping of 4 protons into the mitochondrial intermembrane space.

Pyrazolate-based porphyrinic metal-organic frameworks as catechol oxidase mimic enzyme for fluorescent and colorimetric dual-mode detection of dopamine with high sensitivity and specificity

• A dual-signal readout sensor with good sensitivity and selectivity has been developed for dopamine detection. • The study of catechol oxidase mimicking MOF nanozymes has the potential to expand the range of biomimetic MOFs. • The type of modular used in the synthesis process of MOFs can influence the activity of the final enzyme. Enzyme-simulating nanomaterials, with obvious advantages of high activity, stability and cost-effectiveness, can surrogate the complex natural enzymes and catalyze enzyme-like reactions in various harsh applications. Herein, a novel fluorescent and colorimetric dual-readout sensing platform toward dopamine detection based on pyrazolate-based porphyrinic metal-organic frameworks (MOFs) by coordination regulation strategy to improve the performance of catechol oxidase mimic enzyme activity was constructed. Specifically, BA (benzoic acid) or PA (4-Formyl-1( H )-pyrazole) was introduced as auxiliary ligands to regulate the growth mode of pyrazolate-based porphyrinic MOF materials. H 4 TPP (5,10,15,20-Tetra(1 H -pyrazol-4-yl) porphyrin) and auxiliary ligands coordinated with Cu(II), exposing Cu sites with insufficient coordination and enhancing the interaction with oxygen-active species. Benefiting from the binuclear copper center structure and the preferential adsorption of reactants in the self-reinforced interface, a highly sensitive and selective detection method for dopamine based on Cu-TPP(PA) MOF material was proposed under the intervention of H 2 O 2 . The feasibility of Cu-TPP(PA) MOF in detecting dopamine in human serum samples and as a paper-based analytical device was investigated. This achievement will expand the applications of MOFs in analytical chemistry and open up a new avenue for the design of highly active and stable nano-enzymes.

Histidine Reactive Chemicals Modify the Thermus thermophilus CuA Redox Center

The electron transport chain is a series of redox protein complexes that couple the shuttling of electrons with the pumping of protons, in order to create the electrochemical gradient that drives ATP synthesis. The first redox center of the terminal complex from the Thermus thermophilus bacteria has been isolated as TtCuA and has a binuclear copper center bridged by two cysteines and ligated by two histidines and two weak axial ligands. H157 of TtCuA, a solvent exposed ligating histidine, has demonstrated modification by diethyl pyrocarbonate (DEPC), an exogenous chemical modifier, that is facilitated by the residue’s fast proton exchange with the solvent. The H157‐DEPC adduct is reversible over 48 hours at room temperature, as supported by mass spectrometry, electrochemistry, and visible CD spectroscopy. This is one of two modifications, out of 12, that becomes removed. Due to the lability of the H157‐DEPC bond, it is possible that the H157 Nɛ is primed for proton pumping in the electron transport chain.In order to model a possible in vivo modification, TtCuA was exposed to 4‐hydroxynonenal (HNE), an endogenous lipid peroxidation product associated with multiple metabolic and neurodegenerative diseases. Exposure of TtCuA to 20 equivalents of HNE appears to reduce the protein over 48 hours and four modifications are observed via mass spectrometry. Exposure of the Pseudomonas aeruginosa azurin protein, a mononuclear blue‐copper denitrification protein with similar ligand groups to TtCuA, is not reduced by HNE, despite modification. Azurin also lacks a ligating histidine that can be modified by DEPC. In conclusion, the binuclear copper center of TtCuA has hyperreactivity in comparison to a similarly ligated mononuclear copper center.Support or Funding InformationMurchison Undergraduate Research Fellowship, Trinity University Chemistry Department, Mach Research Fellowship, Welch Foundation

Purple Mixed-Valent Copper A.

CuA is a binuclear copper center acting as an electron transfer hub in terminal oxidases such as cytochrome c oxidase and nitrous oxide reductase. Its unique electronic structure is intimately linked to its function and has puzzled the community of biological inorganic chemistry for decades. Here we review the insights provided by different spectroscopic techniques of CuA centers, and the different experimental approaches to tackle its study, that encompass the synthesis of model compounds as well as protein engineering efforts. The contribution of the electronic structure to the thermodynamic and kinetic of electron transfer is extensively discussed. We also describe the proposed mechanism of CuAassembly in different organisms. The recent discovery of a novel CuA site opens new perspectives to this field.

COA6 Facilitates Cytochrome c Oxidase Biogenesis as Thiol-reductase for Copper Metallochaperones in Mitochondria

The mitochondrial cytochrome c oxidase, the terminal enzyme of the respiratory chain, contains heme and copper centers for electron transfer. The conserved COX2 subunit contains the CuA site, a binuclear copper center. The copper chaperones SCO1, SCO2, and COA6, are required for CuA center formation. Loss of function of these chaperones and the concomitant cytochrome c oxidase deficiency cause severe human disorders. Here we analyzed the molecular function of COA6 and the consequences of COA6 deficiency for mitochondria. Our analyses show that loss of COA6 causes combined complex I and complex IV deficiency and impacts membrane potential-driven protein transport across the inner membrane. We demonstrate that COA6 acts as a thiol-reductase to reduce disulfide bridges of critical cysteine residues in SCO1 and SCO2. Cysteines within the CX3CXNH domain of SCO2 mediate its interaction with COA6 but are dispensable for SCO2–SCO1 interaction. Our analyses define COA6 as thiol-reductase, which is essential for CuA biogenesis.

Carbazole and hydrazone derivatives as new competitive inhibitors of tyrosinase: Experimental clues to binuclear copper active site binding

In this work a total of 12 carbazoles and hydrazone-bridged thiazole-pyrrole derivatives have been identified as new competitive inhibitors of tyrosinase. Carbazole derivative with 2-benzoimidazole substitution showed most potent inhibition in the series. Other carbazole derivatives containing benzothiazole and benzoxazole substitutions showed comparable levels of tyrosinase inhibition. The hydrazone derivatives also showed potent tyrosinase inhibitory activity with comparable Ki values except one with fluoride at its terminal position. Kinetic studies showed competitive inhibition of tyrosinase by all compounds that increased the substrate Km without changing the Vmax value. Moreover, experimental evidence suggests that the target compounds specifically bind to the binuclear copper center of the tyrosinase active site in an apparent mono-dentate fashion. Carbazoles and hydrazones are new and emerging classes of compounds as tyrosinase inhibitors that may provide new structural avenues to discovery of drugs targeting the treatment of hyperpigmentation and related dermatological disorders.

ChemInform Abstract: Tao‐Phos‐Controlled Desymmetrization of Succinimide‐Based Bisalkynes via Asymmetric Copper‐Catalyzed Huisgen Alkyne—Azide Click Cycloaddition: Substrate Scope and Mechanism.

AbstractThe enantioselective 1,3‐dipolar azide cycloaddition affords triazoles with moderate to high enantioselectivity.

Tao-Phos-controlled desymmetrization of succinimide-based bisalkynes via asymmetric copper-catalyzed Huisgen alkyne–azide click cycloaddition: substrate scope and mechanism

It was found that the catalytic Huisgen cycloaddition reaction of succinimide-derived bisalkynes with azides resulted in useful succinimide-derived triazoles bearing quaternary carbon-stereogenic centers with good to excellent yields as well as good chemoselectivity and moderate to high enantioselectivities (up to 97% ee), in which the CuF2/Tao-Phos complex was proved to be an effective bimetallic catalyst in the presence of triethylamine. The mechanistic studies based on the effect of reaction parameters on stereoselectivity as well as ESI-MS analysis suggested that our ligand (Tao-Phos) and related binuclear copper centers play a crucial role in this asymmetric click chemistry because of the strong catalyst–substrate interaction with the aid of a possibly in situ formed binuclear or multinuclear copper-based transition state. This work not only provides a new example of a Cu(II)-mediated asymmetric AAC reaction involving chelating base and azides, but also suggests the significance of binuclear copper species in catalytic asymmetric click reactions with two copper center-involved enantioselective inductions.

Mono-nuclear copper complexes mimicking the intermediates for the binuclear copper center of the subunit II of cytochrome oxidase: a peptide based approach.

Three stable copper complexes of peptides derived from the copper ion binding loop of the subunit II of cytochrome c oxidase have been prepared and characterized by various spectroscopic techniques. These stable copper complexes of peptides were found to exhibit cysteine, histidine and/or methionine ligation, which has predominant σ-contribution in the Cys-Cu charge transfer. The copper(ii) peptide complexes showed type-2 EPR spectra, which is uncommon in copper-cysteinate complexes. UV-visible spectra, Raman and EPR results support a tetragonal structure of the coordination geometry around the copper ion. The copper complex of the 9-amino acid peptide suggested the formation of a 'red' copper center while the copper complexes of the 12- and 11-amino acid peptides showed the formation of a 'green' copper center. The results provide insights on the first stable models of the copper complexes formed in the peptide scaffold that mimic the mono-nuclear copper bound protein intermediates proposed during the formation of the binuclear Cu2S2 core of the enzyme. These three copper complexes of peptides derived from the metal ion binding loop of the CuA center of the subunit II of cytochrome c oxidase showed novel spectroscopic properties which have not so far been reported in any stable small complex.

The pro-enzyme C-terminal processing domain of Pholiota nameko tyrosinase is responsible for folding of the N-terminal catalytic domain.

Pholiota nameko (Pholiota microspore) tyrosinase is expressed as a latent 67-kDa pro-tyrosinase, comprising a 42-kDa N-terminal catalytic domain with a binuclear copper centre and a 25-kDa C-terminal domain and is activated by proteolytic digestion of the C-terminal domain. To investigate the role of the C-terminal processing domain of pro-tyrosinase, we constructed a recombinant tyrosinase lacking the C-terminal domain and four recombinant pro-tyrosinase mutants (F515G, H539N, L540G and Y543G) carrying substituted amino acid residues on the C-terminal domain. The recombinant tyrosinase lacking the C-terminal domain had no catalytic activity; whereas the mutant L540G was copper depleted, the other mutants had copper contents similar to that of the wild-type pro-tyrosinase. Proteolytic digestion activated the mutants H539N and Y543G following release of the C-terminal domain, and the resulting tyrosinases had higher K m values for t-butyl catechol than the wild-type pro-tyrosinase. The mutants F515G and L540G were degraded by proteolytic digestion and yielded smaller proteins with no activity. These data suggest that the C-terminal processing domain of P. nameko pro-tyrosinase is essential for correct folding of the N-terminal catalytic domain and acts as an intramolecular chaperone during assembly of the active-site conformation.

Genome-Wide Analysis of Polyphenol Oxidase Genes and Their Transcriptional Patterns during Grain Development in Sorghum

Polyphenol oxidase (PPO) plays multiple roles in plant growth and responses to environmental stimuli. However, there is still little molecular information on PPO genes in sorghum. Here, eight distinct PPO gene members, designated as SbPPO1–SbPPO8, were identified in two grain sorghum genotypes, i.e., BTx623 and Henong 16 (HN16). The eight members were located on sorghum chromosomes 3 (SbPPO1 and SbPPO2), 6 (SbPPO3 and SbPPO4), 7 (SbPPO5–SbPPO7), or 10 (SbPPO8) and contained zero (SbPPO6–SbPPO8), one (SbPPO1, SbPPO2, and SbPPO5), or two (SbPPO3 and SbPPO4) introns. In BTx623, the deduced proteins of SbPPO1–SbPPO8 all contained an intact binuclear copper center required for enzyme activity, as well as the tyrosine motif and its interacting residues involved in structural maintenance. SbPPO2–SbPPO6 were found to possess thylakoid transfer domains, whereas SbPPO1 and SbPPO8 were predicted to contain N-terminal signal peptides for targeting to the secretory pathway. In HN16, the SbPPO3 allele was a pseudogene, whereas the remaining seven SbPPO alleles showed high identities with their counterparts in BTx623. Phylogenetically, sorghum PPO genes generally exhibited relationships closer to those of maize, and the maize orthologues of SbPPO2–SbPPO8 could be recognized by synteny analysis. Finally, SbPPO5 was found to be the major expressed member at the early stage of grain growth, and the dynamic change of SbPPO5 transcript level paralleled that of the total PPO activity during the grain development in both BTx623 and HN16. Our study generated new information on the PPO members in sorghum, which may facilitate more detailed functional analysis of these important genes in further research.

Direct electron transfer between tyrosinase and multi-walled carbon nanotubes for bioelectrocatalytic oxygen reduction

We report the fabrication of a tyrosinase bioelectrode by mechanical compression of a MWCNT enzyme mixture. Cyclic voltammetry of the nanostructured bioelectrode demonstrated a Direct Electron Transfer (DET) process between tyrosinase, a copper enzyme, and MWCNT. The latter led to an enzyme redox potential of +0.30V vs SCE, close to the redox potential described for the T3 binuclear copper center. Furthermore, we demonstrate, for the first time, a bioelectrocatalytic reduction of oxygen performed by tyrosinase directly wired within the MWCNT disk. A maximum current density of 0.55mAcm−2 was recorded by chronoamperometric measurements at 0V vs SCE. The bioelectrode exhibits excellent stability over time, conserving more than 50% of its activity after one week. DET between MWCNTs and the T3 binuclear copper centers have been further investigated by studying the influence of two tyrosinase inhibitors: benzoic acid and cyanide. As previously reported for “blue” multicopper oxidases, such as bilirubin oxidase and laccase, tyrosinase can achieve oxygen reduction via DET between MWCNT and its T3 binuclear copper center, representing an alternative in the design of oxygen biocathodes for biofuel cells.

Crystallization and Preliminary Analysis of Crystals of the 24-Meric Hemocyanin of the Emperor Scorpion (Pandinus imperator)

Hemocyanins are giant oxygen transport proteins found in the hemolymph of several invertebrate phyla. They constitute giant multimeric molecules whose size range up to that of cell organelles such as ribosomes or even small viruses. Oxygen is reversibly bound by hemocyanins at binuclear copper centers. Subunit interactions within the multisubunit hemocyanin complex lead to diverse allosteric effects such as the highest cooperativity for oxygen binding found in nature. Crystal structures of a native hemocyanin oligomer larger than a hexameric substructure have not been published until now. We report for the first time growth and preliminary analysis of crystals of the 24-meric hemocyanin (MW = 1.8 MDa) of emperor scorpion (Pandinus imperator), which diffract to a resolution of 6.5 Å. The crystals are monoclinc with space group C 1 2 1 and cell dimensions a = 311.61 Å, b = 246.58 Å and c = 251.10 Å (α = 90.00°, β = 90.02°, γ = 90.00°). The asymmetric unit contains one molecule of the 24-meric hemocyanin and the solvent content of the crystals is 56%. A preliminary analysis of the hemocyanin structure reveals that emperor scorpion hemocyanin crystallizes in the same oxygenated conformation, which is also present in solution as previously shown by cryo-EM reconstruction and small angle x-ray scattering experiments.

Chiral one-dimensional hydrogen bonded, antiferromagnetic chloro-bridged dinuclear copper(II) complex with tridentate Schiff base ligand

Chiral one-dimensional hydrogen bonded copper(II) complex [Cu(HL1)Cl]2·H2O (1) (HL1=N-(2-hydroxyethyl)-5-nitrosalicylaldimine) has been synthesized and characterized by elemental analysis, crystal structure analysis and magnetic susceptibility. X-ray diffraction studies show that 1 is a binuclear copper(II) complex with a pair of chlorine atoms bridging the copper atoms in a central Cu2Cl2 core. Each copper(II) atom is five-coordinated in a square-pyramidal environment. Variable-temperature magnetic susceptibility measurements on the a binuclear complex 1 in the range 2–300K indicate a weak antiferromagnetic coupling between the binuclear copper(II) centers, with J=−0.274cm−1 and the magneto-structural correlations are discussed in detail.

A novel type of prophenoloxidase from the kuruma prawn Marsupenaeus japonicus contributes to the melanization of plasma in crustaceans

Melanization is one of the major immune responses in arthropods. Prophenoloxidases (proPOs) catalyze the oxidation of mono- or o-diphenols, a reaction that is the key initial step of melanin formation. Well-characterized proPOs from crustaceans are synthesized in haemocytes and are released into plasma in response to microbial attack. However, PO activity does exist in the plasma of haemolymph without pathogenic infections. Here, we demonstrate that a novel type of proPO contributes to such PO activity in the plasma fraction of haemolymph of crustaceans. The novel enzyme, which was purified from the plasma of the kuruma prawn ( Marsupenaeus japonicus), possessed strong and specific monophenol and o-diphenol oxidation activity compared with that of known haemocyte-type proPO. Amino acid sequence analyses indicated that this enzyme was distinct from the known proPO. The cDNA sequence and deduced amino acid sequence of this enzyme has a putative binuclear copper center, and showed approximately 30% and 20% identity with the primary structures of reported proPO and haemocyanin sequences of the kuruma prawn, respectively. Reverse transcription PCR analysis showed that this enzyme was synthesized in the hepatopancreas rather than in haemocytes. Although the primary structure and enzymatic properties of this novel enzyme suggested that it is a phenoloxidase, its biogenesis, tissue distribution, and oligomeric state resemble those of haemocyanin, which belongs to the same protein family (type III copper protein). This novel proPO enzyme may share a role with the already characterized version, itself a major component of the innate immune system in crustaceans.

Structure–function relationship among bacterial, fungal and plant laccases

Abstract Laccases (benzenediol: oxygen oxidoreductase; EC 1.10.3.2), a multicopper oxidase enzyme, widely distributed in plants, fungi and bacteria have ability to catalyze oxidation of various phenolic and non-phenolic compounds as well as many environmental pollutants. The diversified functions of laccases, including the antagonistic ones such as their involvement in lignin biosynthesis (in plants) as well as lignin degradation (in fungi and bacteria), make them an interesting enzyme for study from the point of view of their structure, function and application. Important applications of laccases include delignification, pulp bleaching and bioremediation. The ability of laccases to polymerize natural phenols helps to develop new cosmetic pigments, hair dyeing materials, deodorants, toothpastes, mouthwashes and other useful products. Recently, the utility of enzyme has also been shown in the nanoparticle based biosensor technology as well as in medical fields. In the present review, a comparative account of the bacterial, fungal and plant laccases has been presented from these points of views. Laccases are dimeric or tetrameric glycoproteins usually containing four copper atoms per monomer. To perform catalytic function, laccase depends on Cu atoms that are distributed at the three different copper centers. These copper centers in laccases are categorized into three groups: Type-1 or blue copper center, Type-2 or normal copper and Type-3 or coupled binuclear copper centers. The four copper atoms are differing in their characteristic electronic paramagnetic resonance (EPR) signals. The phylogenetic analysis reveals, laccases from these groups (viz. bacteria, fungi and plant) form independent clades, in consistent with that of taxonomical classification. From previous experimental evidence and from in silico studies, it is evident that despite their wide taxonomic distribution and substrate diversity, molecular architecture of laccases is common to multicopper oxidases. Three dimensional structure predictions, at monomeric level, for all laccases (bacterial, fungal and plant) suggest that they are composed of three sequentially arranged cupredoxin-like domains. Multiple alignment of primary sequences of all three modeled laccases shows that, the copper binding motives are highly conserved in all sequences. The similarities are more significant in the N- and C-terminal regions, corresponding to Domains 1 and 3, as the copper interacting motif is present in Domains 1 and 3 not in Domain 2. This structural conservation reflects a common reaction mechanism for the copper oxidation and the O2 reduction in these enzymes. Putative binding pocket analysis depicts, larger binding cavity for bacterial laccase as compared to those for plants and fungi. An in depth analysis of copper binding site, yielded significant differences in conserved residues for laccases of bacteria, fungi and plants which provided the basis for the dual and contrasting functions of laccases.

Structural Basis of the Lactate-dependent Allosteric Regulation of Oxygen Binding in Arthropod Hemocyanin

Hemocyanin (Hc) is an oxygen carrier protein in which oxygen binding is regulated by allosteric effectors such as H(+) and L-lactate. Isothermal titration calorimetric measurements showed that L-lactate binds to dodecameric and heterohexameric Hc and to the CaeSS3 homohexamer but not to the CaeSS2 monomer. The binding of lactate caused no change in the optical absorption and x-ray absorption spectra of either oxy- or deoxy-Hc, suggesting that no structural rearrangement of the active site occurred. At pH 6.5, the oxygen binding rate constant k(obs) obtained by flash photolysis showed a significant increase upon addition of L-lactate, whereas L-lactate addition had little effect at pH 8.3. Lactate binding caused a concentration-dependent shift in the interhexameric distances at pH 6.5 based on small angle x-ray scattering measurements. These results show that L-lactate affects oxygen affinity at pH 6.5 by modulating the global structure of Hc without affecting its binuclear copper center (the active site). In contrast to this, the active site structure of deoxy-Hc is affected by changes in pH (Hirota, S., Kawahara, T., Beltramini, M., Di Muro, P., Magliozzo, R. S., Peisach, J., Powers, L. S., Tanaka, N., Nagao, S., and Bubacco, L. (2008) J. Biol. Chem. 283, 31941-31948). Upon addiction of lactate, the kinetic behavior of oxygen rebinding for Hc was heterogeneous under low oxygen concentrations at pH 6.5 due to changes in the T and R state populations, and the equilibrium was found to shift from the T toward the R state with addition of lactate.

Direct electrochemistry of dinuclear CuA fragment from cytochrome c oxidase of Thermus thermophilus at surfactant modified glassy carbon electrode

Abstract Cytochrome c oxidase is ubiquitous enzyme involved in the terminal step of respiratory electron transfer process. The unique binuclear copper center containing bis-dithiolato bridges form a valance delocalized [Cu 1.5+ –Cu 1.5+ ] state of the metal center located at the subunit II of cytochrome c oxidase. This metal center acts as the electron entry site of the enzyme and accepts electrons from cytochrome c. Direct electrochemistry of this binuclear copper center containing the water soluble protein obtained by genetically truncating the membrane bound part of the subunit II from Thermus thermophilus was achieved by favorable orientation of the protein on glassy carbon electrode surface promoting efficient electron transfer in the presence of various surfactants. Very reproducible, Nernstian responses are obtained with Cu A . The redox potential and the electrochemical response were enhanced prominently in case of cationic surfactant CTAB indicating that the nature of the surfactant has a significant effect on the microenvironment of the protein–electrode interface. The results have been used to understand the mechanism of electron transfer from cytochrome c to the copper center during the enzymatic reaction.

CuA centers and their biosynthetic models in azurin

Cu(A) is a binuclear copper center that functions as an electron transfer agent, cycling between a reduced Cu(I)Cu(I) state and an oxidized mixed-valence Cu(+1.5)...Cu(+1.5) state. The copper ions are bridged by two cysteine thiolate ligands and form a copper-copper bond, the first reported of its kind in Nature. Such a "diamond-core" Cu(2)S(Cys)(2) structure allows an unpaired electron to be completely delocalized over the two copper ions and contributes to its highly efficient electron transfer properties. This review provides accounts of how the Cu(A) center was structurally characterized and highlights its salient spectroscopic properties. In the process, it introduces the Cu(A) center in four different systems-native protein systems, soluble protein truncates of native proteins, synthetic models using organic molecules, and biosynthetic models using proteins as ligands-with a greater emphasis on biosynthetic models of Cu(A), especially on new, deeper insights gained from their studies.