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To produce specific antibodies for the detection and quantification of copper ions, bifunctional chelators (BFCs) are commonly applied in the preparation of copper conjugates. However, some copper-chelator complexes exhibit limited stability under in vivo conditions. In this study, Cu 2+ was coupled with carrier proteins via three different macrocyclic BFCs: p-SCN-Bn-DOTA, p-SCN-Bn-NOTA, and p-SCN-Bn-TETA. The stability in plasma and the immunogenicity of three copper immunoconjugates were compared. The chelators other than p-SCN-Bn-DOTA were very stable in plasma, with <9% dissociation of Cu 2+ over 96 h. The immune response varied depending on the choice of chelator; notably, antisera from the Cu 2+ -NOTA-KLH conjugate demonstrated the best reactivity toward chelated Cu 2+ . p-SCN-Bn-NOTA, which showed significant advantages over the other chelators, was used for antibody production. The efficiency of immune-positive hybridoma production was satisfactory, and the resultant monoclonal antibodies (McAbs) 4B7 showed sensitivity (half-maximal inhibitory concentration (IC 50 ) of 8.9 ng/mL) to chelated Cu 2+ , with a working range from 1.21 to 48.9 ng/mL. The recovery of Cu 2+ from water samples was 85.7-108%, and the intra- and inter-assay coefficients of variation were 4.0-10.1% and 7.1-11.4%, respectively. Compared with previously reported McAb specific to Cu 2+ , DF4, the sensitivity of the newly developed assay was improved 100-fold. The results of this study indicate the utility of NOTA for the efficient generation of highly sensitive McAbs against Cu 2+ ., (Copyright © 2019 Elsevier B.V. All rights reserved.)

The redox properties of the μ-η2:η2 peroxo complex [Cu2(H6M4h)(O2)]2+ were elucidated. This study constitutes the first full electrochemical and spectroelectrochemical characterization of a side-on peroxo Cu2:O2 bioinorganic model complex. The peroxo complex is irreversibly reduced in a two-electron process localized on the peroxo ligand triggering the cleavage of the O-O bond.

Monoclonal antibody (MAb) against chelated Cu(2+) was developed. The conjugate employed as immunogens in BALB/c mice to raise antibodies was synthesized using Cu(2+) coupling to keyhole limpet hemocyanin (KLH) via a bifunctional chelator (2-(4-isothiocyanatobenzyl)-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid, p-SCN-Bn-DOTA). One of the hybridomas secreting antibody that bound tightly to Cu(2+)-ethylenediamine tetraacetic acid (EDTA) complex but not to metal-free EDTA was isolated from the fusion between murine splenocytes and SP2/0 myeloma cells. The performance of a competitive enzyme-linked immunosorbent assay (ELISA) was assessed for its sensitivity to changes in pH and ionic strength. The cross-reactivities (CR) with other metals were below 1%, except for Hg(2+) with a CR of 7.19 %. The IC(50) value achieved for copper was 0.89 μg/mL, showing a detection range of 0.25-8.13 μg/mL and the lowest detection limit of 0.032 μg/mL. The concentration of Cu(2+) in environmental water and serum samples obtained by ELISA correlated well with Atomic Absorption Spectrometry (AAS), and the mean recovery was 93.7%. These results indicated that ELISA could be a convenient analytical tool for monitoring copper in drinking water and human serum., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Hemocyanin (Hc) is a type-3 copper protein, containing dioxygen-binding active sites consisting of paired copper atoms. In the present study the thermal unfolding of the Hc from the marine mollusc Rapana thomasiana (RtH) has been investigated by combining differential scanning calorimetry, Fourier transform infrared (FTIR) and UV-vis absorption spectroscopy. Two important stages in the unfolding pathway of the Hc molecule were discerned. A first event, with nonmeasurable heat absorption, occurring around 60°C, lowers the binding of dioxygen to the type-3 copper groups. This pretransition is reversible and is ascribed to a slight change in the tertiary structure. In a second stage, with midpoint around 80°C, the protein irreversibly unfolds with a loss of secondary structure and formation of amorphous aggregates. Experiments with the monomeric structural subunits, RtH1 and RtH2, indicated that the heterogeneity in the process of thermal denaturation can be attributed to the presence of multiple 50kDa functional units with different stability. In accordance, the irreversible unfolding of a purified functional unit (RtH2-e) occurred at a single transition temperature. At slightly alkaline pH (Tris buffer) the C-terminal β-sheet rich domain of the functional unit starts to unfold before the α-helix-rich N-terminal (copper containing) domain, triggering the collapse of the global protein structure. Even around 90°C some secondary structure is preserved as shown by the FTIR spectra of all investigated samples, confirming the high thermostability of molluscan Hc., (Crown Copyright © 2012. Published by Elsevier B.V. All rights reserved.)

Three copper polypyridyl complexes were examined as electrocatalysts for the oxygen reduction reaction (ORR): a Cu-N(3) complex, [Cu-[tris(6-methylpyridin-2-yl)methane]-(NCMe)]PF(6) (1); a related Cu(2)N(6) derivative, [Cu(2)-[1,2-bis(6-(bis(6-methylpyridin-2-yl)methyl)pyridin-2-yl)ethane]-(NCMe)(2)](PF(6))(2) (2); and the CuN(4) species [Cu-[tris(pyridin-2-ylmethyl)amine]](ClO(4))(2) [3](ClO(4))(2). Compared to other copper complexes, [3](ClO(4))(2) exhibits the highest reported ORR onset potential for a Cu complex of 0.53 V vs reversible hydrogen electrode at pH 1. The Cu(2)N(6) hemocyanin model is more active than the CuN(3), but both are less active than the CuN(4) complex. The results indicate that copper polypyridyl complexes are promising cathode catalysts for ORR., (© 2011 American Chemical Society)

The coupled binuclear "type 3" Cu sites are found in hemocyanin (Hc), tyrosinase (Tyr), and the multicopper oxidases (MCOs), such as laccase (Lc), and play vital roles in O(2) respiration. Although all type 3 Cu sites share the same ground state features, those of Hc/Tyr have very different ligand-binding properties relative to those of the MCOs. In particular, the type 3 Cu site in the MCOs (Lc(T3)) is a part of the trinuclear Cu cluster, and if the third (i.e., type 2) Cu is removed, the Lc(T3) site does not react with O(2). Density functional theory calculations indicate that O(2) binding in Hc is approximately 9 kcal mol(-1) more favorable than for Lc(T3). The difference is mostly found in the total energy difference of the deoxy states (approximately 7 kcal mol(-1)), where the stabilization of deoxy Lc(T3) derives from its long equilibrium Cu-Cu distance of approximately 5.5-6.5 A, relative to approximately 4.2 A in deoxy Hc/Tyr. The O(2) binding in Hc is driven by the electrostatic destabilization of the deoxy Hc site, in which the two Cu(I) centers are kept close together by the protein for facile 2-electron reduction of O(2). Alternatively, the lack of O(2) reactivity in Lc(T3) reflects the flexibility of the active site, capable of minimizing the electrostatic repulsion of the 2 Cu(I)s. Thus, the O(2) reactivity of the MCOs is intrinsic to the trinuclear Cu cluster, leading to different O(2) intermediates as required by its function of irreversible reduction of O(2) to H(2)O.

We report the use of triazacyclophane (TAC)-scaffolded amino acids as a structural mimic for 3-histidine metal-binding sites in metalloproteins, especially for the mimicry of type-3 copper binding sites as are present in hemocyanin, tyrosinase and catechol oxidase.

Copper-cluster sites in biology exhibit unique spectroscopic features reflecting exchange coupling between oxidized Cu's and e (-) delocalization in mixed valent sites. These novel electronic structures play critical roles in O 2 binding and activation for electrophilic aromatic attack and H-atom abstraction, the 4e (-)/4H (+) reduction of O 2 to H 2O, and in the 2e (-)/2H (+) reduction of N 2O. These electronic structure/reactivity correlations are summarized below.

The molecular mechanism of the monooxygenase (phenolase) activity of type 3 copper proteins has been examined in detail both in the model systems and in the enzymatic systems. The reaction of a side-on peroxo dicopper(II) model compound ( A) and neutral phenols proceeds via a proton-coupled electron-transfer (PCET) mechanism to generate phenoxyl radical species, which collapse each other to give the corresponding C-C coupling dimer products. In this reaction, a bis(mu-oxo)dicopper(III) complex ( B) generated by O-O bond homolysis of A is suggested to be a real active species. On the other hand, the reaction of lithium phenolates (deprotonated form of phenols) with the same side-on peroxo dicopper(II) complex proceeds via an electrophilic aromatic substitution mechanism to give the oxygenated products (catechols). The mechanistic difference between these two systems has been discussed on the basis of the Marcus theory of electron transfer and Hammett analysis. Mechanistic details of the monooxygenase activity of tyrosinase have also been examined using a simplified enzymatic reaction system to demonstrate that the enzymatic reaction mechanism is virtually the same as that of the model reaction, that is, an electrophilic aromatic substitution mechanism. In addition, the monooxygenase activity of the oxygen carrier protein hemocyanin has been explored for the first time by employing urea as an additive in the reaction system. In this case as well, the ortho-hydroxylation of phenols to catechols has been demonstrated to involve the same ionic mechanism.

Dinuclear [Cu2(mu-O)2(Tp(R,R')2] complexes, analogues of the active site of oxyhemocyanin, are theoretically studied, and the effect of the substituents of the tris(pyrazolyl)borate ligands, Tp(R,R'), is analyzed. Density functional theory calculations reveal that the type of bridging oxygen, peroxo, or bisoxo is strongly influenced by the nature and position of the R substituents because of variable substituent...bridging oxygen interactions, as well as electronic effects. The electronic effects of ligands at the 5 position are not significant, but peroxo complexes are favored by electron-withdrawing groups at the 3 position while bisoxo ones are strongly sterically disfavored.

We have investigated the effect of copper binding on the structural properties of hemocyanin (Hc). To this aim, we have studied the holo- and apo-form of the protein, both in the hexameric and in the monomeric state (CaeSS2 subunit), with experimental approaches that report on the protein aggregation and conformational stability. The results of gel-filtration chromatography and small angle X-ray scattering (SAXS) provide evidence that the hydrodynamic and gyration radius (R(g)) of Hc in the hexameric form only slightly increase upon copper removal, whereas a remarkable enhancement in the R(g) value is observed for the CaeSS2 monomer. CD measurements in the far- and near-UV region indicate that removal of copper only marginally affects the conformation of the hexameric Hc. Instead, copper depletion in the CaeSS2 strongly alters the tertiary structure of the monomer (near-UV CD), even though it is almost inconsequential on the secondary structure content (far-UV CD). These findings are fully consistent with the results of limited proteolysis experiments showing that the hexameric Hc is similarly resistant to proteolysis by trypsin both in the holo- and apo-form. Conversely, the apo-form of CaeSS2 monomer is much more susceptible to proteolytic attack by trypsin than the holo-form. Based on SAXS measurements, the concentration-dependent oligomerization process for apo-CaeSS2 has been analyzed on the basis of a thermodynamic model involving a concentration-dependent equilibrium between a monomer in a native-like and an hexameric aggregate of monomers.

Our previous studies on the met- and met-azido-Hc forms from Octopus vulgaris (mollusc) and Carcinus aestuarii (arthropod) at pH 7.5 and on some related binuclear models without X-ray diffraction have considered and resolved some fundamental aspects of their binuclear type-3 copper site (i.e. correct values of the Cu-Cu distances; apical distortion, when present, at the copper site; presence and type of bridging groups). In this contribution the multiple-scattering (MS) calculations performed in order to refine the EXAFS modulation of the absorption spectra are presented. It is only with a composite and advanced approach that some severe problems, mainly deriving from the presence of two absorbing atoms and from the fact that the metal-metal contribution in the absorption spectra overlaps with the Cu-His signals, have been overcome. Results are also presented which indicate the role of the MS calculations in the XANES edge region, and which show how it is possible to extract quantitative information from this zone of the spectrum in order to refine the structure of the site also in the case of a binuclear centre.

The modelling of the active sites of metalloproteins is one of the most challenging tasks in bio-inorganic chemistry. Copper proteins form part of this stimulating field of research as copper enzymes are mainly involved in oxidation bio-reactions. Thus, the understanding of the structure-function relationship of their active sites will allow the design of effective and environmental friendly oxidation catalysts. This perspective illustrates some outstanding structural and functional synthetic models of the active site of copper proteins, with special attention given to models of galactose oxidase and hemocyanin.

Binuclear Cu proteins play vital roles in O(2) binding and activation in biology and can be classified into coupled and noncoupled binuclear sites based on the magnetic interaction between the two Cu centers. Coupled binuclear Cu proteins include hemocyanin, tyrosinase, and catechol oxidase. These proteins have two Cu centers strongly magnetically coupled through direct bridging ligands that provide a mechanism for the 2-electron reduction of O(2) to a mu-eta(2):eta(2) side-on peroxide bridged Cu(II)(2)(O(2)(2-)) species. This side-on bridged peroxo-Cu(II)(2) species is activated for electrophilic attack on the phenolic ring of substrates. Noncoupled binuclear Cu proteins include peptidylglycine alpha-hydroxylating monooxygenase and dopamine beta-monooxygenase. These proteins have binuclear Cu active sites that are distant, that exhibit no exchange interaction, and that activate O(2) at a single Cu center to generate a reactive Cu(II)/O(2) species for H-atom abstraction from the C-H bond of substrates. O(2) intermediates in the coupled binuclear Cu enzymes can be trapped and studied spectroscopically. Possible intermediates in noncoupled binuclear Cu proteins can be defined through correlation to mononuclear Cu(II)/O(2) model complexes. The different intermediates in these two classes of binuclear Cu proteins exhibit different reactivities that correlate with their different electronic structures and exchange coupling interactions between the binuclear Cu centers. These studies provide insight into the role of exchange coupling between the Cu centers in their reaction mechanisms.

In this work we show, by a combination of biochemical and biophysical approaches, that the copper ions bound in the binuclear active site of Carcinus aestuarii hemocyanin play a stabilizing role on the tertiary structure of the protein. Upon removal of copper, the monomeric hemocyanin, but not the hexameric oligomer, undergoes changes at the level of tertiary structure while the secondary structure is almost unaffected. By Small-Angle X-Ray Scattering, supported by gel chromatography measurements, it can be concluded that the apo-monomer, but not the holo form or the hexameric form, undergoes a slow time-dependent oligomerization process.

The structural basis that define the physiological functions of binuclear copper enzymes is discussed in the frame of the data generated by a broad spectroscopic approach, spanning from paramagnetic NMR and pulsed EPR to x-ray absorption spectroscopies. The structural features discussed for the different oxidation and ligation states accessible to a binuclear copper sites are the coordination geometry for the first and second shell, the metal-metal distance and the role of the bridging exogenous ligand(s). A structural model will be presented to rationalize both the differentiation in function within the protein families and the reaction mechanism of those proteins that are enzymatically active.

The preferred state, the peroxide Cu(2)(II,II) or the bis-mu-oxo Cu(2)(III,III) states, for oxygen-bridged copper dimers with nitrogen donors is reinvestigated. Experiments have indicated that for the enzymatic complexes with histidine ligands the peroxide state should be favored, at least for hemocyanin, while for the synthetic complexes with strained ligands the bis-mu-xo state should be intrinsically favored. The present B3LYP study essentially agrees with these results. The quite different results obtained in CASPT2 and some previous B3LYP studies for these systems are investigated and discussed. The conclusion, drawn in an earlier study, that the Cu(2)(III,III) state is an unlikely intermediate in the enzyme mechanisms of tyrosinase and catechol oxidase, still remains.

The crystal structure of catechol oxidase reveals new insight into the functional properties of the type-3 copper proteins. This class of proteins includes the closely related and better-known tyrosinase as well as hemocyanin, an oxygen transport protein. All these proteins have a dinuclear copper center, have similar spectroscopic behaviors, and show close evolutionary and functional relationships. Comparison between the 3D structures of catechol oxidase and hemocyanins reveals the structural reasons for the divergence in function.

We have identified three MT encoding genes in the blue crab: MT-I, inducible by cadmium, zinc and copper; MT-II, inducible by cadmium and zinc; and MT-III, inducible by copper only [Syring et al., Comp. Biochem. Physiol. C, 125 (2000) 325-332]. To examine the role of the CuMT-I and CuMT-III isoforms in copper metabolism associated with the synthesis and degradation of the oxygen-binding copper protein, hemocyanin, we (1) cloned and sequenced hemocyanin cDNA, (2) examined interaction of the CuMTs with endoplasmic reticulum (ER) vesicles and (3) measured changes in levels of hemocyanin, MT-I, MT-III protein and mRNA that occur in crabs during different stages of the molt cycle. The cDNA-derived hemocyanin amino-acid sequence revealed the presence of a leader peptide indicating that hemocyanin is a secretory protein that is synthesized on the ER. Copper uptake studies show that ER vesicles take up both Cu1+ and Cu2+ in an ATP-independent process. The copper transporter has a Km of 10.8+/-2.4 microM copper and a Vmax of 6.1+/-0.5 nmol Cu/mg protein/10 min. ER vesicles contain hemocyanin, and bind CuMT-I and, preferentially, CuMT-III. However, binding does not result in copper transfer to the ER. There are statistically significant changes in hepatopancreas MT-III and hemocyanin mRNA, and in hemolymph hemocyanin concentrations during the molt cycle. MT-I mRNA remains constant. Changes in MT-III mRNA are positively correlated with changes in hemocyanin mRNA and hemocyanin protein, which points to coordinate control of MT-III and hemocyanin transcription. No CuMT-III protein is observed in hepatopancreas of intermolt crabs when levels of both MT-III and hemocyanin mRNA are high, suggesting rapid utilization of copper bound to MT-III when cells are actively synthesizing hemocyanin. CuMT-III is present in premolt and softshell crabs, and its emergence appears to coincide with a decrease in hemocyanin synthesis and increase in hemocyanin degradation. These results support the hypothesis that the copper-specific metallothionein is intricately involved in copper homeostasis associated with both the synthesis and degradation of hemocyanin.

The structural characteristics of oxy- and deoxy-hemocyanins have been investigated using X-ray absorption spectroscopy both in the near-edge (XANES) and for the first shell contribution in the EXAFS region. Several arthropodan and molluscan hemocyanins have been studied in order to trace the inter- and intra-phyla differences. The XANES spectra of oxy-hemocyanins of the different species are remarkably similar, consistent with a very strongly conserved co-ordination geometry of the copper active site. In contrast, small but significant differences are observed between the deoxy-forms of arthropodan and molluscan proteins. In particular, the XANES spectra of deoxy-arthropodan hemocyanins (with the exception of L. polyphemus Hc) show a more intense edge feature at approximately 8983 eV. This difference is tentatively assigned to a more planar geometry of the copper-ligands system in the arthropodan rather than in the molluscan proteins. The first shell analysis of the EXAFS modulation is consistent with the presence of n=3Nepsilon(2) imidazole nitrogens at an average distance of 1.92 +/- 0.03 A from copper in all the deoxy-hemocyanins investigated. Binding of dioxygen results for all hemocyanins in the increase of the number of first shell back-scattering atoms to n=5 with average distances of 1.93 A. Alternatively, by separating the contribution of Nepsilon(2) imidazole nitrogens and of peroxide O-atoms, n=3 ligands at 1.98 +/- 0.03 A and n=2 ligands at 1.87 +/- 0.03 A are found.

Two closely related copper proteins, phenoloxidase and haemocyanin, are known to be involved in different physiological functions such as the primary immune response and oxygen transport. Although the proteins differ structurally, they have the same active site by which dioxygen is bound. Recent results reveal that haemocyanin also exhibits phenoloxidase activity. A scenario is proposed for the evolutionary relationships among copper oxygen-binding proteins (COPs).

Nitric oxide (NO) can act as a ligand for copper atoms and may also engage in redox chemistry with the metal once bound. Furthermore NO posses an unpaired electron which can couple with the unpaired electron on Cu2+. These properties have been exploited to probe the active sites of copper-containing enzymes and proteins. We review these studies. In addition to the use as a spectroscopic probe for the active site we draw attention to the rapid reactions of NO at the copper sites in Cytochrome c oxidase (CcO) and laccase. These reactions in CcO occur in the ms time range, at low NO concentrations and in the presence of oxygen and may therefore be of physiological relevance to the control of respiration. Finally we speculate on the wider role that NO may play in regulation of an important group of Type 2 copper containing enzymes.

We purified two catechol oxidases from Lycopus europaeus and Populus nigra which only catalyze the oxidation of catechols to quinones without hydroxylating tyrosine. The molecular mass of the Lycopus enzyme was determined to 39,800 Da and the mass of the Populus enzyme was determined to 56,050 Da. Both catechol oxidases are inhibited by thiourea, N-phenylthiourea, dithiocarbamate, and cyanide, but show different pH behavior using catechol as substrate. Atomic absorption spectrosopic analysis found 1.5 copper atoms per protein molecule. Using EPR spectroscopy we determined 1.8 Cu per molecule catechol oxidase. Furthermore, EPR spectroscopy demonstrated that catechol oxidase is a copper enzyme of type 3. The lack of an EPR signal is due to strong antiferromagnetic coupling that requires a bridging ligand between the two copper ions in the met preparation. Addition to H2O2 to both enzymes leads to oxy catechol oxidase. In the UV/Vis spectrum two new absorption bands occur at 345 nm and 580 nm. In accordance with the oxy forms of hemocyanin and tyrosinase the absorption band at 345 nm is due to an O2(2-) (pi sigma *)-->Cu(II) (dx2 - y2) charge transfer (CT) transition. The absorption band at 580 nm corresponds to the second O2(2)- (pi v*)-->Cu(II) (dx2 - y2) CT transition. The UV/Vis bands in combination with the resonance Raman spectra of oxy catechol oxidase indicate a mu-eta 2:eta 2 binding mode for dioxygen. The intense resonance Raman peak at 277 cm-1, belonging to a Cu-N (axial His) stretching mode, suggests that catechol oxidase has six terminal His ligands, as known for molluscan and arthropodan hemocyanin.

Catechol oxidases are ubiquitous plant enzymes containing a dinuclear copper center. In the wound-response mechanism of the plant they catalyze the oxidation of a broad range of ortho-diphenols to the corresponding o-quinones coupled with the reduction of oxygen to water. The crystal structures of the enzyme from sweet potato in the resting dicupric Cu(II)-Cu(II) state, the reduced dicuprous Cu(I)-Cu(I) form, and in complex with the inhibitor phenylthiourea were analyzed. The catalytic copper center is accommodated in a central four-helix-bundle located in a hydrophobic pocket close to the surface. Both metal binding sites are composed of three histidine ligands. His 109, ligated to the CuA site, is covalently linked to Cys 92 by an unusual thioether bond. Based on biochemical, spectroscopic and the presented structural data, a catalytical mechanism is proposed in which one of the oxygen atoms of the diphenolic substrate binds to CuB of the oxygenated enzyme.

Mono-azide adduct of Rhus vernicifera laccase, a multicopper oxidase containing one type-1 (blue) copper, one type-2 (non-blue normal) copper, and a pair of type-3 (binuclear and EPR silent) coppers, of which type-2 and type-3 coppers constitute a trinuclear site, was investigated with resonance Raman (RR) and Fourier transform infrared (FT-IR) spectroscopies as a step toward elucidation of the structure and function of the trinuclear site. The Cu-N3- stretching (vCu-N3-) RR band was observed for azide-bound multicopper oxidases for the first time. The vCu-N3- band was located at 400 cm-1 for mono-14N3- laccase, which shifted to 396 cm-1 with the 15N14N14N3- analog. The N3- asymmetric stretching (v(N3-)asym) band was observed by FT-IR spectroscopy at 2035 cm-1 for mono-14N3- laccase and at 2025 cm-1 for the 15N14N14N3- analog. The vCu-N3- and v(N3-)asym frequencies and their 15N14N14N- isotope shifts for azido laccase correspond well with those of metazido hemocyanin, indicating that both derivatives should have a similar binding geometry of azide.

To establish the competence of the active site of hemocyanin to acquire diverse coordination geometries, the binding of azide to three forms of a crab hemocyanin, the dinuclear cupric or met-hemocyanin, the mononuclear cupric or met-apo-hemocyanin, and the mononuclear Co(II)-substituted derivative has been studied by near-ultraviolet circular dichroism and EPR spectroscopies. The near-ultraviolet circular dichroism spectra of the various derivatives present qualitatively similar features, namely a negative peak around 335 nm in the case of the two copper-containing derivatives and a three-component pattern with the Co(II) derivative. Upon decreasing the pH from 7.0 to 5.5 a decrease of optical activity is observed with all protein samples. The characteristic CD features, attributable to N(imidazole)-to-metal and to OH -to-metal charge-transfer transitions, are strongly affected by azide binding. In particular, the intensity of the negative band exhibited by the two copper-containing protein forms decreases with the onset of a new negative feature with maximum around 400 nm diagnostic for azide-to-Cu(II) charge-transfer transitions. The visible region is affected as well, indicating that changes in the coordination sphere of copper take place. The affinity for azide of the different protein forms is higher at low pH. EPR measurements on the paramagnetic met-apo-hemocyanin derivative as a function of pH demonstrate heterogeneity in the coordination environment at low pH. In the presence of azide an increase of rhombic distortion of the EPR spectra is observed and on the basis of the identified sets of copper hyperfine features in the course of azide titration experiments two different azide bound forms of met-apo-hemocyanin can be detected. The CD and EPR data at the different pH values are consistent with a reaction scheme in which azide replaces a fourth ligand in the metal-coordination sphere, identified as a water or hydroxide molecule.

We have measured the x-ray absorption near edge structure (XANES) spectra of the enzyme tyrosinase from the mushroom Agaricus bisporus in solution in the oxy and deoxy forms. The spectra, obtained under the same conditions as the analogous forms of mollusc hemocyanin (Hc), show that the oxidation state of copper changes from Cu(II) (oxy form) to Cu(I) (deoxy form), and the copper active site(s) of A. bisporus tyrosinase in solution undergoes the same main conformational changes as Hc. We have applied the multiple scattering theory to simulate the XANES spectra of various alternative geometries of the copper site, accounting for the residual differences between Hc and tyrosinase. While oxy-Hc is reasonably fitted only by the pseudo-square-pyramidal geometry reported by its crystallographic data, oxytyrosinase can be fitted, starting from the Hc coordinates, either by distortions toward a pseudo-tetrahedral geometry, with inequivalent copper sites, or by an apically distorted square-pyramidal geometry (with an elongation of the apical distance of no more than 0.2 A).

Pro-phenol oxidase [pro-PO; zymogen of phenol oxidase (monophenol, L-dopa:oxygen oxidoreductase, EC 1.14.18.1)] is present in the hemolymph plasma of the silkworm Bombyx mori. Pro-PO is a heterodimeric protein synthesized by hemocytes. A specific serine proteinase activates both subunits through a limited proteolysis. The amino acid sequences of both subunits were deduced from their respective cDNAs; amino acid sequence homology between the subunits was 51%. The deduced amino acid sequences revealed domains highly homologous to the copper-binding site sequences (copper-binding sites A and B) of arthropod hemocyanins. The overall sequence homology between silkworm pro-PO and arthropod hemocyanins ranged from 29 to 39%. Phenol oxidases from prokaryotes, fungi, and vertebrates have sequences homologous to only the copper-binding site B of arthropod hemocyanins. Thus, silkworm pro-PO DNA described here appears distinctive and more closely related to arthropod hemocyanins. The pro-PO-activating serine proteinase was shown to hydrolyze peptide bonds at the carboxyl side of arginine in the sequence-Asn-49-Arg-50-Phe-51-Gly-52- of both subunits. Amino groups of N termini of both subunits were indicated to be N-acetylated. The cDNAs of both pro-PO subunits lacked signal peptide sequences. This result supports our contention that mature pro-PO accumulates in the cytoplasm of hemocytes and is released by cell rupture, as for arthropod hemocyanins.

Hemocyanin (Hc) is a dinuclear copper protein that binds oxygen reversibly. The structure of the Cu(II) site in a derivative of hemocyanin known as green half-met (GHM) has been analyzed using the pulsed EPR technique of electron spin-echo envelope modulation (ESEEM) spectroscopy. The derivative, prepared by treating the native protein with nitrite at low pH, contains a mixed-valent binuclear copper center. It was shown through chemical assays and the ligand exchange reaction products identified by EPR spectroscopy to contain a nitrite ligand bound to Cu(II). The ESEEM spectra of green half-methemocyanins from mollusks and arthropods indicated that three imidazole ligands are coordinated to Cu(II). Therefore, a tetragonal N3O ligand structure (O is an oxygen of nitrite) is proposed. For GHM Hc from the mollusks Octopus vulgaris and Rapana thomasiana, the isotropic nitrogen nuclear hyperfine coupling constant, aiso, for the N delta (or remote) nitrogen of two imidazoles was approximately 1.4 MHz, while for the third, aiso congruent to 2.2 MHz. The difference between the two weaker nitrogens and the single, more strongly coupled nitrogen was smaller by 0.2 MHz in the GHM Hcs from the arthropods Carcinus maenas, Homarus americanus and Panulirus interruptus. The nitrogen nuclear quadrupole coupling constants and asymmetry parameters, e2Qq and eta, for the N delta nitrogens in nearly all cases were near 1.4 MHz and 0.8, respectively, although Rapana thomasiana GHM Hc exhibited a reduction in eta that may indicate weaker hydrogen bonding in the active site of this protein. The g and ACu (copper nuclear hyperfine coupling) values for the derivatives, and the finding of three similar nuclear hyperfine coupling constants for the N delta sites of imidazole ligands, when considered with the orientation-specific information obtained using angle-selection methods for simulation of ESEEM spectra, suggest a distorted tetragonal Cu(II) structure in which three imidazoles and a nitrite ligand are bound near the equatorial plane. The finding that the two molluscan GHM Hcs exhibit differences associated with the remote nitrogen of imidazoles bound to Cu(II) may be related to a structural variability in the active sites of these proteins not found in the arthropodan GHM Hcs examined.

The preparation of a mononuclear Cu(II) derivative of Carcinus maenas hemocyanin (Cu(II)-Hc) and a nitrite complex of the derivative (Cu(II)-Hc-NO2-) are described. Several techniques have been used in their characterization, including X-ray absorption, continuous wave (cw) EPR, and electron spin-echo envelope modulation (ESEEM) spectroscopies. EXAFS results for Cu(II)-Hc indicate the presence of three ligands at 1.99 +/- 0.01 A and a fourth one at 2.26 +/- 0.01 A from the copper. The same coordination number and very similar bond lengths were obtained for Cu(II)-Hc-NO2-. On the basis of simulations of three-pulse ESEEM spectra, three equivalent imidazole nitrogens coupled to Cu(II) were identified in Cu(II)-Hc. Upon the binding of nitrite, a decrease in the hyperfine interaction for two of the three imidazole nitrogens was observed by ESSEM. Further, the results of a two-pulse ESEEM experiment are consistent with the assignment of the protons of a water ligand to Cu(II), which is displaced when nitrite is added. An analysis of X-ray absorption K-edge spectra suggests a coordination geometry intermediate between square-planar and tetrahedral for the metal centers in Cu(II)-Hc and Cu(II)-Hc-NO2-, in agreement with the g and ACu values determined by cw-EPR. On the basis of these results, an equivalent structure is suggested for Cu(II)-Hc-NO2- and the Cu(II) site in green half-methemocyanin, a partially oxidized binuclear derivative formed in the reaction of the native protein with nitrite.