Biochemical characterization of CopA,the Escherichia coli Cu(I)-translocating P-type ATPase

Escherichia coli CopA is a copper ion-translocating P-type ATPase that confers copper resistance. CopA formed a phosphorylated intermediate with [gamma-(32)P]ATP. Phosphorylation was inhibited by vanadate and sensitive to KOH and hydroxylamine, consistent with acylphosphate formation on conserved Asp-523. Phosphorylation required a monovalent cation, either Cu(I) or Ag(I). Divalent cations Cu(II), Zn(II), or Co(II) could not substitute, signifying that the substrate of this copper-translocating P-type ATPase is Cu(I) and not Cu(II). CopA purified from dodecylmaltoside-solubilized membranes similarly exhibited Cu(I)/Ag(I)-stimulated ATPase activity, with a K(m) for ATP of 0.5 mm. CopA has two N-terminal Cys(X)(2)Cys sequences, Gly-Leu-Ser-Cys(14)-Gly-His-Cys(17), and Gly-Met-Ser-Cys(110)-Ala-Ser-Cys(113), and a Cys(479)-Pro-Cys(481) motif in membrane-spanning segment six. The requirement of these cysteine residues was investigated by the effect of mutations and deletions. Mutants with substitutions of the N-terminal cysteines or deletion of the first Cys-(X)(2)-Cys motif formed acylphosphate intermediates. From the copper dependence of phosphoenzyme formation, the mutants appear to have 2-3 fold higher affinity for Cu(I) than wild type CopA. In contrast, substitutions in Cys(479) or Cys(481) resulted in loss of copper resistance, transport and phosphoenzyme formation. These results imply that the cysteine residues of the Cys-Pro-Cys motif (but not the N-terminal cysteine residues) are required for CopA function.     

Beta-endorphin. Synthesis and biological activity of analogs with disulfide bridges

Two analogs of human beta-endorphin (beta-EP) which contain cystine bridges, [Cys15-Cys26,Phe27,Gly31]-beta-EP (I) and [Cys16-Cys26,Phe27,Gly31]-beta-EP (II), were synthesized by the solid-phase method. Peptides I and II were shown to contain 2-2.5 times the opiate receptor binding activity of beta-endorphin. We also synthesized two analogs with reduced alkylated cysteine residues and these peptides, [Arg9,19,24,28,29 Cys(Cam)11,26,Phe27,Gly31] and [Arg9,19,24,28,29,Cys-(Cam)12,26,Phe27,Gly31], were shown to have approximately the same opiate receptor activity as beta-endorphin.     

The computed distribution of copper(II) and zinc(II) ions among seventeen amino acids present in human blood plasma

The equilibrium distribution of copper(II) and zinc(II) ions among a mixture of 17 amino acids has been computed from stability-constant and blood-plasma-composition data. At pH7.4, 98% of the copper(II) in the simulated plasma solution is co-ordinated to histidine and cystine, predominantly as the mixed-ligand complexes [Cu.His.Cystine](-) and [Cu.H.His.Cystine]. Approximately half of the zinc(II) is co-ordinated to cysteine and histidine, but appreciable complex-formation occurs with most of the other amino acids. Stability constants are given for copper(II) and zinc(II) amino acid complexes, including some mixed-ligand species, at 37 degrees C and I=0.15m.     

Mechanistic flexibility in the reduction of copper(II) complexes of aliphatic polyamines by mercapto amino acids

The reactions of copper(II)-aliphatic polyamine complexes with cysteine, cysteine methyl ester, penicillamine, and glutathione have been investigated, with the goal of understanding the relationship between RS- -Cu(II) adduct structure and preferred redox decay pathway. Considerable mechanistic flexibility exists within this class of mercapto amino acid oxidations, as changes in the rate law could be induced by modest variations in reductant concentration (at fixed [Cu(II)]0), pH, and the structure of the redox partners. With excess cysteine present at 25 degrees C, pH 5.0, I = 0.2 M (NaOAc), decay of 1:1 cys-S- -Cu(II) transient adducts was found to be first order in both cys-SH and transient. Second-order rate constants characteristic of Cu(dien)2+(6.1 X 10(3) M-1 sec-1), Cu(Me5dien)2+ (2.7 X 10(3) M-1 sec-1), Cu(en)22+ (2.1 X 10(3) M-1 sec-1), and Cu(dien)22+ (4.7 X 10(3) M-1 sec-1) are remarkably similar, considering substantial differences in the composition and geometry of the oxidant first coordination sphere. A mechanism involving attack of cysteine on the coordinated sulfur atom of the transient, giving a disulfide anion radical intermediate, is proposed to account for these results. Moderate reactivity decreases in the cysteine-Cu(dien)2+, Cu(Me5dien)2+ reactions with increasing [H+] (pH 4-6) reflect partial protonation of the polyamine ligands. A very different rate law, second order in the RS- -Cu(II) transient and approximately zeroth order in mercaptan, applies in the pH 5.0 oxidations of cysteine methyl ester, penicillamine, and glutathione by Cu(dien)2+ and Cu(Me5dien)2+. This behavior suggests the intermediacy of di-mu-mercapto-bridged binuclear Cu(II) species, in which a concerted two-electron change yields the disulfide and Cu(I) products. Similar hydroxo-bridged intermediates are proposed to account for the transition from first- to second-order transient dependence in cysteine oxidations by Cu(dien)2+ and Cu(Me5dien)2+ as the pH is increased from 5 to 7. Yet another rate law, second order in transient and first order in cysteine, applies in the pH 5.0 oxidation of cysteine by Cu(Me6tren)2+ (k(25 degrees C) 7.5 X 10(7) M-2 sec-1, I = 0.2M). Steric rigidity of this trigonal bipyramidal oxidant evidently protects the coordinated sulfur atom from attack in a RSSR- -forming pathway. Formation of a coordinated disulfide in the rate-determining step is proposed, coupled with attack of a noncoordinated cysteine molecule on a vacated coordination position to stabilize the (Me6tren)Cu(I) product.     

Localization of the intrachain disulfide bonds of the envelope glycoprotein 71 from Friend murine leukemia virus.

Envelope glycoprotein 71 from Friend murine leukemia virus was purified to homogeneity by reversed-phase HPLC. It could be shown that all 20 cysteine residues of the molecule are linked by disulfide bonds. After complete tryptic digestion, peptides containing cystine were identified by comparison of the reversed-phase HPLC profile of the digest with that of a reduced aliquot which had been subjected to affinity chromatography on thiol-Sepharose. The locations of the 10 disulfide bonds were determined by isolation, further digestion and analysis of peptides containing cystine. The first cysteine residue of the sequence (Cys46) was shown to be coupled to the sixth (Cys98), leading to a large loop containing four additional cysteine residues. Computer model building and energy calculations led to the assignment of Cys72 to Cys87 and Cys73 to Cys83. The following four cysteine residues of the sequence also constitute a structural unit, with Cys121 bonded to Cys141 and Cys133 to Cys146, and the last two cysteine residues in the amino-terminal domain of glycoprotein 71 form a small loop (Cys178 to Cys184). The first two cysteine residues of the carboxy-terminal domain produce a very small hydrophobic loop (Cys312-Cys315). Cys361 is bound to Cys373, Cys342 to Cys396 and Cys403 to Cys416. A model for the folding pattern of the viral glycoprotein is proposed.     

Ternary Cu(II) complexes, Cu(H(-1)L)(ACys-) and Cu(H(-2)L)(ACys-); L = peptides, ACys- = N-acetyl-cysteinate. Analogous complexes to the intermediates in the transport of Cu(II) from Cu(H(-2)L) to cysteine

The Cu(II) in Cu(H(-2)L) has been postulated to be successively transported to cysteine (Cys) as follows; Cu(H(-2)L) <==> Cu(H(-2)L)(Cys*-) <==> Cu(H(-1)L)(Cys*-) --> Cu(H(-1)L)(Cys-), where Cys*- denotes the monodentate Cys-. N-acetyl-cysteinate (ACys-) complexes Cu(H(-2)L)(ACys-) and Cu(H(-1)L)(ACys-), having similar coordination modes to Cu(H(-2)L)(Cys*-) and Cu(H(-1)L)(Cys*-), respectively, exhibited the S --> Cu(II) charge transfer absorption at 325-355 nm and the d-d absorption at 530-610 nm. A linear interrelation existed between the energies of the CD and d-d absorptions. Cu(H(-2)L)(ACys-) were in rapid equilibrium with Cu(H(-1)L)(ACys-). Upon forming the ternary complex, pK(c2) of the parent Cu(H(-1)L) was raised to more than 1.0. The formation constants (K) of the Cu(H(-1)L)(ACys-) species from Cu(H(-1)L) were bigger than those of Cu(H(-2)L)(ACys-) from Cu(H(-2)L). The linear free-energy relationship existed between the free-energy change (deltaG) and the entropy change (deltaS) for the ternary complex formation. The rate constants (k1+) for the Cu(H(-1)L)(Cys-) formation closely correlated with the K values for Cu(H(-2)L)(ACys-). The ternary complexes containing ACys are considered to be analogous complexes to the intermediates in the transport of Cu(II) from peptides to cysteine.     

A model of the copper centres of nitrous oxide reductase (Pseudomonas stutzeri). Evidence from optical, EPR and MCD spectroscopy.

Nitrous oxide reductase (N2OR), Pseudomonas stutzeri, catalyses the 2 electron reduction of nitrous oxide to di-nitrogen. The enzyme has 2 identical subunits (Mr approximately 70,000) of known amino acid sequence and contains approximately 4 Cu ions per subunit. By measurement of the optical absorption, electron paramagnetic resonance (EPR) and low-temperature magnetic circular dichroism (MCD) spectra of the oxidised state, a semi-reduced form and the fully reduced state of the enzyme it is shown that the enzyme contains 2 distinct copper centres of which one is assigned to an electron-transfer function, centre A, and the other to a catalytic site, centre Z. The latter is a binuclear copper centre with at least 1 cysteine ligand and cycles between oxidation levels Cu(II)/Cu(II) and Cu(II)/Cu(I) in the absence of substrate or inhibitors. The state Cu(II)/Cu(I) is enzymatically inactive. The MCD spectra provide evidence for a second form of centre Z, which may be enzymatically active, in the oxidised state of the enzyme. Centre A is structurally similar to that of CuA in bovine and bacterial cytochrome c oxidase and also contains copper ligated by cysteine. This centre may also be a binuclear copper complex.     

Structure of the complex of Streptomyces griseus proteinase B and polypeptide chymotrypsin inhibitor-1 from Russet Burbank potato tubers at 2.1 A resolution

A low molecular weight protein inhibitor of serine proteinases from Russet Burbank potato tubers, polypeptide chymotrypsin inhibitor-1 (PCI-1), has been crystallized in complex with Streptomyces griseus proteinase B (SGPB). The three-dimensional structure of the complex has been solved at 2.1 A resolution by the molecular replacement method and has been refined to a final R-factor (= sigma[[Fo[-[Fc[[/sigma[Fo[) of 0.142 (8.0 to 2.1 A resolution data). The reactive site bond of PCI-1 (Leu38I to Asn39I) is intact in the complex, and there is no significant distortion of the peptide from planarity. The distance between the active site serine O gamma of SGPB and the carbonyl carbon of the scissile bond of PCI-1 is 2.8 A (1 A = 0.1 nm). The inhibitor has little secondary structure, having a three-stranded antiparallel beta-sheet on the side opposite the reactive site and four beta-turns. PCI-1 has four disulphide bridges; these presumably take the place of extensive secondary structure in keeping the reactive site conformationally constrained. The pairing of the cystine residues, which had not been characterized chemically, is as follows: Cys3I to Cys40I, Cys6I to Cys24I, Cys7I to Cys36I, and Cys13I to Cys49I. The molecular structure of SGPB in the PCI-1 complex agrees closely with the structure of SGPB complexed with the third domain of the turkey ovomucoid inhibitor (OMTKY3). A least-squares overlap of all atoms in SGPB gives a root-mean-square difference of 0.37 A. One of the loops of SGPB (Ser35 to Gly40) differs in conformation in the two complexes by more than 2.0 A root-mean-square for the main-chain atoms. Thr39 displays the largest differences with the carbonyl carbon atom deviating by 3.6 A. This conformational alternative is a result of the differences in the molecular structures of the P'4 residues following the reactive site bonds of the two inhibitors. This displacement avoids a close contact (1.3 A) between the carbonyl oxygen of Ser38 of SGPB and Pro42I C beta of PCI-1. The solvent structure of the PCI-1-SGPB complex includes 179 waters, two sulphate or phosphate ions, and one calcium or potassium ion, which appears to play a role in crystal formation. The molecular structure of PCI-1 determined here has allowed the proposal of a model for the structure of a two-domain inhibitor from potatoes and tomatoes, inhibitor II.(ABSTRACT TRUNCATED AT 400 WORDS)     

X-ray absorption studies of the copper-beta domain of rat liver metallothionein

Rat liver metallothionein contains two domains, each of which enfolds a separate metal-thiolate cluster. The binding stoichiometry of these clusters depends on the particular metal ion bound. In the aminoterminal beta domain the cluster can accommodate either three Cd(II) ions or six Cu(I) ions. The Cd ions are known to be coordinated in a tetrahedral geometry. In order to better understand the binding of Cu ions in this domain, the Cu-beta domain fragment of metallothionein was prepared and investigated by x-ray absorption spectroscopy. Quantitative analysis of the EXAFS data indicates copper-sulfur distances of 2.25 +/- 0.03 A. The EXAFS amplitudes and distance results are most consistent with trigonal coordination. A trigonal biprism is proposed for the Cu6Cys9 complex in which Cu occupies each vertex and cysteinyl sulfur bridges at each of the nine edges.     

Soft metal ions, Cd(II) and Hg(II), induce triple-stranded alpha-helical assembly and folding of a de novo designed peptide in their trigonal geometries

We previously reported the de novo design of an amphiphilic peptide [YGG(IEKKIEA)4] that forms a native-like, parallel triple-stranded coiled coil. Starting from this peptide, we sought to regulate the assembly of the peptide by a metal ion. The replacement of the Ile18 and Ile22 residues with Ala and Cys residues, respectively, in the hydrophobic positions disrupted of the triple-stranded alpha-helix structure. The addition of Cd(II), however, resulted in the reconstitution of the triple-stranded alpha-helix bundle, as revealed by circular dichroism (CD) spectroscopy and sedimentation equilibrium analysis. By titration with metal ions and monitoring the change in the intensity of the CD spectra at 222 nm, the dissociation constant Kd was determined to be 1.5 +/- 0.8 microM for Cd(II). The triple-stranded complex formed by the 113Cd(II) ion showed a single 113Cd NMR resonance at 572 ppm whose chemical shift was not affected by the presence of Cl- ions. The 113Cd NMR resonance was connected with the betaH protons of the cysteine residue by 1H-113Cd heteronuclear multiple quantum correlation spectroscopy. These NMR results indicate that the three cysteine residues are coordinated to the cadmium ion in a trigonal-planar complex. Hg(II) also induced the assembly of the peptide into a triple-stranded alpha-helical bundle below the Hg(II)/peptide ratio of 1/3. With excess Hg(II), however, the alpha-helicity of the peptide was decreased, with the change of the Hg(II) coordination state from three to two. Combining this construct with other functional domains should facilitate the production of artificial proteins with functions controlled by metal ions.     

Penicillamine deprotonations and interactions with copper ions

In aqueous solutions about five times as many penicillamine molecules possess ionized sulfhydryl groups as deprotonated amino groups. That the corresponding ratio for cysteine is two is reaffirmed. N-Acetyl-dl-penicillamine and d-penicillamine react with Cu(II), even in acid solutions, to yield the corresponding disulfide and Cu(I). Cu(I) binds on the average only one sulfhydryl molecule, and a polymeric structure is suggested. A mixed valence state purple species absorbing at 520 nm and stable even in air is formed when approximately equivalent amounts of Cu(I), Cu(II), and d-penicillamine are present in neutral solutions. In the absence of oxygen and in the presence of 0.1 n base d-penicillamine forms a 2:1 complex with Cu(II) that is stable for hours. Absorption and circular dichroism are reported for the above species and the Cu(II) complexes of l-cystinediamide and l-cystinylbisglycine.     

The mitochondrial copper metallochaperone Cox17 exists as an oligomeric, polycopper complex

Cox17 is the candidate copper metallochaperone for delivery of copper ions to the mitochondrion for assembly of cytochrome c oxidase. Cox17 purified as a recombinant molecule lacking any purification tag binds three Cu(I) ions per monomer in a polycopper cluster as shown by X-ray absorption spectroscopy. The CuCox17 complex exists in a dimer/tetramer equilibrium with a 20 microM k(d). The spectroscopic data do not discern whether the dimeric complex forms a single hexanuclear Cu(I) cluster or two separate trinuclear Cu(I) clusters. The Cu(I) cluster(s) exhibit(s) predominantly trigonal Cu(I) coordination. The cluster(s) in Cox17 resemble(s) the polycopper clusters in Ace1 and the Cup1 metallothionein in being pH-stable and luminescent. The physical properties of the CuCox17 complex purified as an untagged molecule differ from those reported previously for a GST-Cox17 fusion protein. The CuCox17 cluster is distinct from the polycopper cluster in Cup1 in being labile to ligand exchange. CuCox17 localized within the intermitochondrial membrane space appears to be predominantly tetrameric, whereas the cytosolic CuCox17 is primarily a dimeric species. Cys-->Ser substitutions at Cys23, Cys24, or Cys26 abolish the Cox17 function and prevent tetramerization, although Cu(I) binding is largely unaffected. Thus, the oligomeric state of Cox17 may be important to its physiological function.     

Mutational analysis of the mitochondrial copper metallochaperone Cox17

The copper metallochaperone Cox17 is proposed to shuttle Cu(I) ions to the mitochondrion for the assembly of cytochrome c oxidase. The Cu(I) ions are liganded by cysteinyl thiolates. Mutational analysis on the yeast Cox17 reveals three of the seven cysteinyl residues to be critical for Cox17 function, and these three residues are present in a Cys-Cys-Xaa-Cys sequence motif. Single substitution of any of these three cysteines with serines results in a nonfunctional cytochrome oxidase complex. Cells harboring such a mutation fail to grow on nonfermentable carbon sources and have no cytochrome c oxidase activity in isolated mitochondria. Wild-type Cox17 purified as untagged protein binds three Cu(I) ions/molecule. Mutant proteins lacking only one of these critical Cys residues retain the ability to bind three Cu(I) ions and are imported within the mitochondria. In contrast, Cox17 molecules with a double Cys --> Ser mutation exhibit no Cu(I) binding but are still localized to the mitochondria. Thus, mitochondrial uptake of Cox17 is not restricted to the Cu(I) conformer of Cox17. COX17 was originally cloned by virtue of complementation of a mutant containing a nonfunctional Cys --> Tyr substitution at codon 57. The mutant C57Y Cox17 fails to accumulate within the mitochondria but retains the ability to bind three Cu(I) ions. A C57S Cox17 variant is functional, and a quadruple Cox17 mutant with C16S/C36S/C47S/C57S substitutions binds three Cu(I) ions. Thus, only three cysteinyl residues are important for the ligation of three Cu(I) ions. A novel mode of Cu(I) binding is predicted.     

Cysteine-to-serine mutants of the human copper chaperone for superoxide dismutase reveal a copper cluster at a domain III dimer interface

Cysteine-to-serine mutants of a maltose binding protein fusion with the human copper chaperone for superoxide dismutase (hCCS) were studied with respect to (i) their ability to transfer Cu to E,Zn superoxide dismutase (SOD) and (ii) their Zn and Cu binding and X-ray absorption spectroscopic (XAS) properties. Previous work has established that Cu(I) binds to four cysteine residues, two of which, C22 and C25, reside within an Atox1-like N-terminal domain (DI) and two of which, C244 and C246, reside in a short unstructured polypeptide chain at the C-terminus (DIII). The wild-type (WT) protein shows an extended X-ray absorption fine structure (EXAFS) spectrum characteristic of cluster formation, but it is not known how such a cluster is formed. Cys to Ser mutagenesis was used to investigate the Cu binding in more detail. Single Cys to Ser mutations, as represented by C22S and C244S, did little to affect the metal binding ratios of hCCS. Both mutants still showed approximately 2 Cu(I) ions and 1 Zn ion per protein. The double mutants C22/24S and C244/246S, on the other hand, showed Cu binding stoichiometries close to 1:1. The Zn-EXAFS of WT CCS showed a 3-4 histidine ligand environment that is consistent with Zn binding in the SOD-like domain II of CCS. The Zn environment remained unchanged between wild type and all of the mutant CCS proteins. Single Cys to Ser mutations displayed lower activity than WT protein, although close to full activity could be rescued by increasing the CCS:SOD ratios to 8:1 in the assay mixture. The structure of the Cu centers of the single mutants as revealed by EXAFS was also similar to that of WT protein, with clear indications of a Cu cluster. On the other hand, the double mutants showed a greater degree of perturbation. The DI C22/25S mutant was 70% active and formed a cluster with a more intense Cu-Cu interaction. The DIII C244/246S mutant retained only a fraction (16%) of activity and did not form a cluster. The results suggest the formation of a DIII-DIII cluster within a dimeric or tetrameric protein and further suggest that this cluster may be an important element of the copper transfer machinery.     

Differential affinity of BsSCO for Cu(II) and Cu(I) suggests a redox role in copper transfer to the Cu(A) center of cytochrome c oxidase

SCO (synthesis of cytochrome c oxidase) proteins are involved in the assembly of the respiratory chain enzyme cytochrome c oxidase acting to assist in the assembly of the Cu(A) center contained within subunit II of the oxidase complex. The Cu(A) center receives electrons from the reductive substrate ferrocytochrome c, and passes them on to the cytochrome a center. Cytochrome a feeds electrons to the oxygen reaction site composed of cytochrome a(3) and Cu(B). Cu(A) consists of two copper ions positioned within bonding distance and ligated by two histidine side chains, one methionine, a backbone carbonyl and two bridging cysteine residues. The complex structure and redox capacity of Cu(A) present a potential assembly challenge. SCO proteins are members of the thioredoxin family which led to the early suggestion of a disulfide exchange function for SCO in Cu(A) assembly, whereas the copper binding capacity of the Bacillus subtilis version of SCO (i.e., BsSCO) suggests a direct role for SCO proteins in copper transfer. We have characterized redox and copper exchange properties of apo- and metalated-BsSCO. The release of copper (II) from its complex with BsSCO is best achieved by reducing it to Cu(I). We propose a mechanism involving both disulfide and copper exchange between BsSCO and the apo-Cu(A) site. This article is part of a Special Issue entitled: Biogenesis/Assembly of Respiratory Enzyme Complexes.     

Mass-spectrometric characterization of two posttranslational modifications of cysteine dioxygenase

Recent crystal structures of cysteine dioxygenase (CDO) suggest the presence of two posttranslational modifications adjacent to the catalytic iron center: a thioether cross-link between Cys93 and Tyr157 and extra electron density at Cys164 which was variously explained as cystine or cysteine sulfinic acid. Purification of recombinant rat CDO yields “mature” and “immature” forms with distinct electrophoretic mobilities. We have positively identified and characterized the two modifications in the products of three sequential proteolytic digestions using liquid chromatography coupled with tandem mass spectrometry. The cross-link is unique to the mature form and was identified in an ion of m/z 3,225.403, consistent with a Tyr-Cys cross-link of peptides Gly80-Phe94 with His155-Phe167. The cross-link is liable to cleavage by in-source decay and the resulting separate peptides were sequenced by collision-induced dissociation tandem mass spectrometry. Mass-spectrometric analysis of these same and overlapping peptides in the presence or absence of reductants and alkylating agents identified the second modification to be a cystine formed between Cys164 and exogenous cysteine as proposed earlier. Both modifications have been shown to form in the presence of high levels of cysteine and iron. This and the presence of small amounts of an apparently off-pathway cystine at position Cys93 suggest that although these conditions promote CDO maturation, they may actually arise via nonenzymatic, nonphysiological processes. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Synthesis and characterisation of anthracene-based fluorophore and its interactions with selected metal ions

An anthracene-based novel ligand (L), 9,10-bis((4,6-dimethylpyrimidin-2-ylthio)methyl)anthracene, was synthesised and fully characterised. Interactions of the ligand with selected metal ions, Hg(II), Cu(II), Ag(I), Pb(II), Zn(II), Ni(II), Co(II), and Cr(III), were spectroscopically investigated. Of the examined metal ions, both Hg(II) and Cu(II) showed responses in both UV-Vis and fluorescent spectroscopy towards the ligand in acetonitrile solution. Spectroscopic titration indicated that the ligand forms complexes with the two metal ions in 1:1 and 1:2 ratios, respectively. DFT calculations revealed that Hg(II) binds possibly with two pairs of donor-set {SN} of the ligand to form a mononuclear complex in a distorted planar geometry whereas Cu(II) forms likely a binuclear complex in a tetrahedral geometry in which each Cu(II) is further coordinated with possibly two acetonitrile molecules.     

Spectral, kinetic, and thermodynamic properties of Cu(I) and Cu(II) binding by methanobactin from Methylosinus trichosporium OB3b

To examine the potential role of methanobactin (mb) as the extracellular component of a copper acquisition system in Methylosinus trichosporium OB3b, the metal binding properties of mb were examined. Spectral (UV-visible, fluorescence, and circular dichroism), kinetic, and thermodynamic data suggested copper coordination changes at different Cu(II):mb ratios. Mb appeared to initially bind Cu(II) as a homodimer with a comparatively high copper affinity at Cu(II):mb ratios below 0.2, with a binding constant (K) greater than that of EDTA (log K = 18.8) and an approximate DeltaG degrees of -47 kcal/mol. At Cu(II):mb ratios between 0.2 and 0.45, the K dropped to (2.6 +/- 0.46) x 10(8) with a DeltaG degrees of -11.46 kcal/mol followed by another K of (1.40 +/- 0.21) x 10(6) and a DeltaG degrees of -8.38 kcal/mol at Cu(II):mb ratios of 0.45-0.85. The kinetic and spectral changes also suggested Cu(II) was initially coordinated to the 4-thiocarbonyl-5-hydroxy imidazolate (THI) and possibly Tyr, followed by reduction to Cu(I), and then coordination of Cu(I) to 4-hydroxy-5-thiocarbonyl imidazolate (HTI) resulting in the final coordination of Cu(I) by THI and HTI. The rate constant (k(obsI)) of binding of Cu(II) to THI exceeded that of the stopped flow apparatus that was used, i.e., >640 s(-)(1), whereas the coordination of copper to HTI showed a 6-8 ms lag time followed by a k(obsII) of 121 +/- 9 s(-)(1). Mb also solubilized and bound Cu(I) with a k(obsI) to THI of >640 s(-)(1), but with a slower rate constant to HTI (k(obsII) = 8.27 +/- 0.16 s(-)(1)), and appeared to initially bind Cu(I) as a monomer.     

Bathocuproine-assisted reduction of copper(II) by human albumin

Human albumin (studied here as the recombinant protein rHA), a copper-binding protein in blood plasma, is shown to reduce Cu(II) to Cu(I) in the presence of a Cu(I) chelator, bathocuproinedisulfonate (BD). This reaction was accelerated at low pH, when there was little binding of Cu(II) to rHA. The addition of a competitive metal ion, Ni(II), or an increase in the concentration of BD, enhanced the reduction of Cu(II) to Cu(I). It was concluded that the oxidant was the Cu(II) complex of BD, which is likely to bind strongly to albumin. The free thiol at Cys34 was ruled out as the sole reducing agent, since Cys34-blocked albumin also gave rise to Cu(I) in the presence of BD. Reactions with amino acids and peptides suggested that Tyr and possibly His side-chains are potential reductants. BD and its homologues are frequently used as Cu(I)-specific chelators in biological experiments, but the strong oxidant activity of [Cu(II)(BD)2]2- and its ability to bind to biological macromolecules should not be overlooked, and may artificially trigger/accelerate Cu(II) reduction.