The Human Amyloid Precursor Protein Binds Copper Ions Dominated by a Picomolar-Affinity Site in the Helix-Rich E2 Domain.

A manifestation of Alzheimer's disease (AD) is the aggregation in the brain of amyloid ß (Aß) peptides derived from the amyloid precursor protein (APP). APP has been linked to modulation of normal copper homeostasis, while dysregulation of Aß production and clearance has been associated with disruption of copper balance. However, quantitative copper chemistry on APP is lacking, in contrast to the plethora of copper chemistry available for Aß peptides. The soluble extracellular protein domain sAPPα (molar mass including post-translational modifications of ∼100 kDa) has now been isolated in good yield and high quality. It is known to feature several copper binding sites with different affinities. However, under Cu-limiting conditions, it binds either Cu(I) or Cu(II) with picomolar affinity at a single site (labeled M1) that is located within the APP E2 subdomain. M1 in E2 was identified previously by X-ray crystallography as a Cu(II) site that features four histidine side chains (H313, H386, H432, and H436) as ligands. The presence of CuII(His)4 is confirmed in solution at pH ≤7.4, while Cu(I) binding involves either the same ligands or a subset. The binding affinities are pH-dependent, and the picomolar affinities for both Cu(I) and Cu(II) at pH 7.4 indicate that either oxidation state may be accessible under physiological conditions. Redox activity was observed in the presence of an electron donor (ascorbate) and acceptor (dioxygen). A critical analysis of the potential biological implications of these findings is presented.

Reduction potentials of protein disulfides and catalysis of glutathionylation and deglutathionylation by glutaredoxin enzymes.

Glutaredoxins (Grxs) are a class of GSH (glutathione)-dependent thiol-disulfide oxidoreductase enzymes. They use the cellular redox buffer GSSG (glutathione disulfide)/GSH directly to catalyze these exchange reactions. Grxs feature dithiol active sites and can shuttle rapidly between three oxidation states, namely dithiol Grx(SH)2, mixed disulfide Grx(SH)(SSG) and oxidized disulfide Grx(SS). Each is characterized by a distinct standard reduction potential [Formula: see text] The [Formula: see text] values for the redox couple Grx(SS)/Grx(SH)2 are available, but a recent estimate differs by over 100 mV from the literature values. No estimates are available for [Formula: see text] for the mixed disulfide couple Grx(SH)(SSG)/(Grx(SH)2 + GSH). This work determined both [Formula: see text] and [Formula: see text] for two representative Grx enzymes, Homo sapiens HsGrx1 and Escherichia coli EcGrx1. The empirical approaches were verified rigorously to overcome the sensitivity of these redox-labile enzymes to experimental conditions. The classic method of acid 'quenching' was demonstrated to shift the thiol-disulfide redox equilibria. Both enzymes exhibit an [Formula: see text] (vs. SHE) at a pH of 7.0. Their [Formula: see text] values (-213 and -230 mV for EcGrx1 and HsGrx1, respectively) are slightly less negative than that ([Formula: see text]) of the redox buffer GSSG/2GSH. Both [Formula: see text] and [Formula: see text] vary with log [GSH], but the former more sensitively by a factor of 2. This confers dual catalytic functions to a Grx enzyme as either an oxidase at low [GSH] or as a reductase at high [GSH]. Consequently, these enzymes can participate efficiently in either glutathionylation or deglutathionylation. The catalysis is demonstrated to proceed via a monothiol ping-pong mechanism relying on a single Cys residue only in the dithiol active site.

C-F bond activation of trifluoroethanol and trifluoroacetic acid catalysed by the dimolybdate anion, [Mo2O6(F)]- †.

Two gas-phase catalytic cycles involving C-F bond activation of trifluoroethanol and trifluoroacetic acid were detected by multistage mass spectrometry experiments. A binuclear dimolybdate centre [Mo2O6(F)]- acts as the catalyst in each cycle. The first cycle, entered via the reaction of [Mo2O6(OH)]- with trifluoroethanol and elimination of water to form [Mo2O6(OCH2CF3)]-, proceeds via four steps: (1) oxidation of the alkoxo ligand and its elimination as aldehyde; (2) reaction of [Mo2O5(OH)]- with trifluoroethanol and elimination of water to form [Mo2O5(OCH2CF3)]; (3) decomposition of the alkoxo ligand via loss of 1,1 difluoroethene; and (4) reaction of [Mo2O6(F)]- with a second equivalent of trifluoroethanol to regenerate Mo2O6(OCH2CF3)]-. Steps (2) and (3) do not occur at room temperature and require collisional activation to proceed. The second cycle is entered via the reaction of [Mo2O6(OH)]- with trifluoroacetic acid and elimination of water to form [Mo2O6(O2CCF3)]- and involves two steps only: (1) fluoride transfer to a molybdenum centre to form [Mo2O6(F)]-; (2) reaction of [Mo2O6(F)]- with trifluoroacetic acid and loss of water to regenerate [Mo2O6(O2CCF3)]-. Comparisons are made with the chemistry of [Mo2O6(OH)]- reacting with acetic acid.

Copper ATPase CopA from Escherichia coli: Quantitative Correlation between ATPase Activity and Vectorial Copper Transport.

Cu-ATPases are membrane copper transporters present in all kingdoms of life. They play a central role in Cu homeostasis by pumping Cu ions across cell membranes with energy derived from ATP hydrolysis. In this work, the Cu-ATPase CopA from Escherichia coli was expressed and purified in fully functional form and demonstrated to bind Cu(I) with subfemtomolar affinity. It was incorporated into the lipid membrane of giant unilamellar vesicles (GUVs) whose dimensions match those of eukaryotic cells. An 1H NMR approach provided a quantitative ATPase activity assay for the enzyme either dissolved in detergent or embedded in GUV membranes. The activity varied with the Cu(I) availability in an optimized assay solution for either environment, demonstrating a direct correlation between ATPase activity and Cu(I) transport. Quantitative analysis of the Cu content trapped by the GUVs is consistent with a Cu:ATP turnover ratio of 1.

Molecular Aspects of a Robust Assay for Ferroxidase Function of Ceruloplasmin.

Ceruloplasmin (Cp) is one of the most complex multicopper oxidase enzymes and plays an essential role in the metabolism of iron in mammals. Ferrous ion supplied by the ferroportin exporter is converted by Cp to ferric ion that is accepted by plasma metallo-chaperone transferrin. Study of the enzyme at the atomic and molecular level has been hampered by the lack of a suitable ferrous substrate. We have developed the classic chromophoric complex FeIIHx(Tar)2 (H2Tar, 4-(2-thiazolylazo)resorcinol; x = 0-2; overall charge omitted) as a robust substrate for evaluation of the ferroxidase function of Cp and related enzymes. The catalysis can be followed conveniently in real time by monitoring the solution absorbance at 720 nm, a fingerprint of FeIIHx(Tar)2. The complex is oxidized to its ferric form FeIIIHx(Tar)2 via the overall reaction sequence FeIIHx(Tar)2 → FeII-Cp → FeIII-Cp → FeIIIHx(Tar)2: i.e., Fe(II) is transferred formally from FeIIHx(Tar)2 to the substrate docking/oxidation (SDO) site(s) in Cp, followed by oxidation to product Fe(III) that is trapped again by the ligand. Each Tar ligand in the above bis-complex coordinates the metal center in a meridional tridentate mode involving a pH-sensitive -OH group (pKa > 12), and this imposes rapid Fe(II) and Fe(III) transfer kinetics to facilitate the catalytic process. The formation constants of both the ferrous and ferric complexes at pH 7.0 were determined (log ß2' = 13.6 and 21.6, respectively), as well as an average dissociation constant of the SDO site(s) in Cp (log KD' = -7.2).

CopC protein from Pseudomonas fluorescens SBW25 features a conserved novel high-affinity Cu(II) binding site.

Copper homeostasis in the bacterium Pseudomonas fluorescens SBW25 appears to be mediated mainly via chromosomal cue and cop systems. Under elevated copper levels that induce stress, the cue system is activated for expression of a P1B-type ATPase to remove excess copper from the cytosol. Under copper-limiting conditions, the cop system is activated to express two copper uptake proteins, Pf-CopCD, to import this essential nutrient. Pf-CopC is a periplasmic copper chaperone that may donate copper to the inner membrane transporter Pf-CopD for active copper importation. A database search revealed that Pf-CopC belongs to a new family of CopC proteins (designated Type B in this work) that differs significantly from the known CopC proteins of Type A that possess two separated binding sites specific for Cu(I) and Cu(II). This article reports the isolation and characterization of Pf-CopC and demonstrates that it lacks a Cu(I) binding site and possesses a novel Cu(II) site that binds Cu(II) with 100 times stronger affinity than do the Type A proteins. Presumably, this is a requirement for a copper uptake role under copper-limiting conditions. The Cu(II) site incorporates a highly conserved amino terminal copper and nickel (ATCUN) binding motif, NH2-Xxx-Xxx-His, but the anticipated ATCUN binding mode is prevented by a thermodynamically more favorable binding mode comprising His1 as a key bidentate ligand and His3 and His85 as co-ligands. However, upon His1 mutation, the ATCUN binding mode is adopted. This work demonstrates how a copper chaperone may fine tune its copper binding site to meet new challenges to its function.

Photochemical oxidation of water and reduction of polyoxometalate anions at interfaces of water with ionic liquids or diethylether.

Photoreduction of [P(2)W(18)O(62)](6-), [S(2)Mo(18)O(62)](4-), and [S(2)W(18)O(62)](4-) polyoxometalate anions (POMs) and oxidation of water occurs when water-ionic liquid and water-diethylether interfaces are irradiated with white light (275-750 nm) or sunlight. The ionic liquids (ILs) employed were aprotic ([Bmim]X; Bmim = (1-butyl-3-methylimidazolium, X = BF(4), PF(6)) and protic (DEAS = diethanolamine hydrogen sulphate; DEAP = diethanolamine hydrogen phosphate). Photochemical formation of reduced POMs at both thermodynamically stable and unstable water-IL interfaces led to their initial diffusion into the aqueous phase and subsequent extraction into the IL phase. The mass transport was monitored visually by color change and by steady-state voltammetry at microelectrodes placed near the interface and in the bulk solution phases. However, no diffusion into the organic phase was observed when [P(2)W(18)O(62)](6-) was photo-reduced at the water-diethylether interface. In all cases, water acted as the electron donor to give the overall process: 4POM + 2H(2)O + hν → 4POM(-) + 4H(+) + O(2). However, more highly reduced POM species are likely to be generated as intermediates. The rate of diffusion of photo-generated POM(-) was dependent on the initial concentration of oxidized POM and the viscosity of the IL (or mixed phase system produced in cases in which the interface is thermodynamically unstable). In the water-DEAS system, the evolution of dioxygen was monitored in situ in the aqueous phase by using a Clark-type oxygen sensor. Differences in the structures of bulk and interfacial water are implicated in the activation of water. An analogous series of reactions occurred upon irradiation of solid POM salts in the presence of water vapor.

Mo-Cu metal cluster formation and binding in an orange protein isolated from Desulfovibrio gigas.

The orange protein (ORP) isolated from the sulfate-reducing bacterium Desulfovibrio gigas (11.8 kDa) contains a mixed-metal sulfide cluster of the type [S2MoS2CuS2MoS2](3-) noncovalently bound to the polypeptide chain. The D. gigas ORP was heterologously produced in Escherichia coli in the apo form. Different strategies were used to reconstitute the metal cluster into apo-ORP and obtain insights into the metal cluster synthesis: (1) incorporation of a synthesized inorganic analogue of the native metal cluster and (2) the in situ synthesis of the metal cluster on the addition to apo-ORP of copper chloride and tetrathiomolybdate or tetrathiotungstate. This latter procedure was successful, and the visible spectrum of the Mo-Cu reconstituted ORP is identical to the one reported for the native protein with absorption maxima at 340 and 480 nm. The (1)H-(15)N heteronuclear single quantum coherence spectra of the reconstituted ORP obtained by strategy 2, in contrast to strategy 1, exhibited large changes, which required sequential assignment in order to identify, by chemical shift differences, the residues affected by the incorporation of the cluster, which is stabilized inside the protein by both electrostatic and hydrophobic interactions.

Unification of the copper(I) binding affinities of the metallo-chaperones Atx1, Atox1, and related proteins: detection probes and affinity standards.

Literature estimates of metal-protein affinities are widely scattered for many systems, as highlighted by the class of metallo-chaperone proteins, which includes human Atox1. The discrepancies may be attributed to unreliable detection probes and/or inconsistent affinity standards. In this study, application of the four Cu(I) ligand probes bicinchoninate, bathocuproine disulfonate, dithiothreitol (Dtt), and glutathione (GSH) is reviewed, and their Cu(I) affinities are re-estimated and unified. Excess bicinchoninate or bathocuproine disulfonate reacts with Cu(I) to yield distinct 1:2 chromatophoric complexes [Cu(I)L(2)](3-) with formation constants ß(2) = 10(17.2) and 10(19.8) m(-2), respectively. These constants do not depend on proton concentration for pH ≥7.0. Consequently, they are a pair of complementary and stable probes capable of detecting free Cu(+) concentrations from 10(-12) to 10(-19) m. Dtt binds Cu(I) with K(D) ∼10(-15) m at pH 7, but it is air-sensitive, and its Cu(I) affinity varies with pH. The Cu(I) binding properties of Atox1 and related proteins (including the fifth and sixth domains at the N terminus of the Wilson protein ATP7B) were assessed with these probes. The results demonstrate the following: (i) their use permits the stoichiometry of high affinity Cu(I) binding and the individual quantitative affinities (K(D) values) to be determined reliably via noncompetitive and competitive reactions, respectively; (ii) the scattered literature values are unified by using reliable probes on a unified scale; and (iii) Atox1-type proteins bind Cu(I) with sub-femtomolar affinities, consistent with tight control of labile Cu(+) concentrations in living cells.

Molecular basis of the cooperative binding of Cu(I) and Cu(II) to the CopK protein from Cupriavidus metallidurans CH34.

The bacterium Cupriavidus metallidurans CH34 is resistant to high environmental concentrations of many metal ions. Upon copper challenge, it upregulates the periplasmic protein CopK (8.3 kDa). The function of CopK in the copper resistance response is ill-defined, but CopK demonstrates an intriguing cooperativity: occupation of a high-affinity Cu(I) binding site generates a high-affinity Cu(II) binding site, and the high-affinity Cu(II) binding enhances Cu(I) binding. Native CopK and targeted variants were examined by chromatographic, spectroscopic, and X-ray crystallographic probes. Structures of two distinct forms of Cu(I)Cu(II)-CopK were defined, and structural changes associated with occupation of the Cu(II) site were demonstrated. In solution, monomeric Cu(I)Cu(II)-CopK features the previously elucidated Cu(I) site in Cu(I)-CopK, formed from four S(δ) atoms of Met28, -38, -44, and -54 (site 4S). Binding of Cu(I) to apo-CopK induces a conformational change that releases the C-terminal ß-strand from the ß-sandwich structure. In turn, this allows His70 and N-terminal residues to form a large loop that includes the Cu(II) binding site. In crystals, a polymeric form of Cu(I)Cu(II)-CopK displays a Cu(I) site defined by the S(δ) atoms of Met26, -38, and -54 (site 3S) and an exogenous ligand (modeled as H(2)O) and a Cu(II) site that bridges dimeric CopK molecules. The 3S Cu(I) binding mode observed in crystals was demonstrated in solution in protein variant M44L where site 4S is disabled. The intriguing copper binding chemistry of CopK provides molecular insight into Cu(I) transfer processes. The adaptable nature of the Cu(I) coordination sphere in methionine-rich clusters allows copper to be relayed between clusters during transport across membranes in molecular pumps such as CusA and Ctr1.

Ionic liquid-enhanced photooxidation of water using the polyoxometalate anion [P2W18O62](6-) as the sensitizer.

Simple polyoxometalate anions are known to be photoreduced in molecular solvents in the presence of 2-propanol or benzyl alcohol. The use of ionic liquids (ILs) as the solvent is now reported to also allow the photooxidation of water to be achieved. In particular, the photochemistry of the classic Dawson polyoxometalate salt K(6)[P(2)W(18)O(62)] has been studied in detail when water is present in the aprotic IL, 1-butyl-3-methylimidazolium tetrafluoroborate ([Bmim][BF(4)]) and the protic IL, diethanolamine hydrogen sulfate (DEAS). In these and other ILs, irradiation with white light (wavelength 275-750 nm) or UV light (wavelength 275-320 nm) leads to overall reduction of the [P(2)W(18)O(62)](6-) anion to [P(2)W(18)O(62)](7-) and concomitant oxidation of water to dioxygen and protons. The modified structure of bulk water present in ILs appears to facilitate its oxidation. Analogous results were obtained in aqueous solutions containing the protic IL as an electrolyte. The photoproducts (reduced polyoxometalate anion, dioxygen, and protons) were identified by, respectively, voltammetry, a Clark electrode, and monitoring of pH. The formal reversible potentials E(0)(F) for [P(2)W(18)O(62)](6-/7-/8-/9-/10-) couples are much more positive than in molecular solvents. The [P(2)W(18)O(62)](8-) and more reduced anions, if formed as intermediates, would efficiently reduce photoproducts H(+) or dioxygen to produce [P(2)W(18)O(62)](7-), rather than reform to [P(2)W(18)O(62)](6-). Thus, under photoirradiation conditions [P(2)W(18)O(62)](7-) acts as a kinetic sink so that in principle indirect splitting of water to produce dioxygen and dihydrogen can be achieved. The equivalent form of photooxidation does not occur in liquid water or in molecular solvents such as MeCN and MeCN/CH(2)Cl(2) containing added water, but does occur for solid K(6)[P(2)W(18)O(62)] in contact with water vapor.

Reaction mechanisms of the multicopper oxidase CueO from Escherichia coli support its functional role as a cuprous oxidase.

CueO from Escherichia coli is a multicopper oxidase (MCO) involved in copper tolerance under aerobic conditions. It features four copper atoms that act as electron transfer (T1) and dioxygen reduction (T2, T3; trinuclear) sites. In addition, it displays a methionine-rich insert which includes a helix that blocks physical access to the T1 site and which provides an extra labile site T4 adjacent to the T1 center. This T4 site is required for CueO function. Like many MCOs, CueO exhibits phenol oxidase activity with broad substrate specificity. Maximal activity with model substrate 2,6-dimethoxyphenol required stoichiometric occupation of T4 by Cu(II) (notation: Cu(II)-CueO). This was achieved in Mops buffer which has little affinity for Cu(2+). However, pH buffers that bind or precipitate Cu(2+) (Tris, BisTris, and KPi) generated enzyme with a vacant T4 site (notation: square-CueO) which has no phenol oxidase activity. Addition of excess Cu(2+) effectively generated a Cu(2+) buffer and recovered the activity partially or completely, depending upon the specific pH buffer. This phenomenon allowed reliable estimation of the affinity of T4 for Cu(II): K(D) = 5.5 x 10(-9) M. CueO is involved in copper tolerance and has been suggested to be a cuprous oxidase. The anion [Cu(I)(Bca)(2)](3-) (Bca = bicinchoninate) acted as a novel chromophoric substrate. It is a robust reagent, being air-stable and having a Cu(I) affinity comparable to those of periplasmic Cu(I) binding proteins. The influences of pH buffer composition and of excess Cu(2+) on cuprous oxidation were diametrically opposite to those seen for phenol oxidation, suggesting that square-CueO, not Cu(II)-CueO, is the resting form of the cuprous oxidase. Steady-state kinetics demonstrated that the intact anion [Cu(I)(Bca)(2)](3-), not "free" Cu(+), is the substrate that donates Cu(I) directly to T4. The data did not follow classical Michaelis-Menten kinetics but could be fitted satisfactorily by an extension that considered the effect of free ligand Bca. The K(m) term consists of two components, allowing estimation of the transient affinity of T4 for Cu(I): K(D) = 1.3 x 10(-13) M. It may be concluded that Cu(I) carried by [Cu(I)(Bca)(2)](3-) is oxidized only upon complete transfer of Cu(I) to T4. The transfer is required to induce a negative shift in the copper reduction potential to allow oxidation and electron transfer to the T1 site. The results provide compelling evidence that CueO is a cuprous oxidase. The new approach will have significant application to the study of metallo-oxidase enzymes.

Solvation effects on S K-edge XAS spectra of Fe-S proteins: normal and inverse effects on WT and mutant rubredoxin.

S K-edge X-ray absorption spectroscopy (XAS) was performed on wild type Cp rubredoxin and its Cys --> Ser mutants in both solution and lyophilized forms. For wild type rubredoxin and for the mutants where an interior cysteine residue (C6 or C39) is substituted by serine, a normal solvent effect is observed, that is, the S covalency increases upon lyophilization. For the mutants where a solvent accessible surface cysteine residue is substituted by serine, the S covalency decreases upon lyophilization which is an inverse solvent effect. Density functional theory (DFT) calculations reproduce these experimental results and show that the normal solvent effect reflects the covalency decrease due to solvent H-bonding to the surface thiolates and that the inverse solvent effect results from the covalency compensation from the interior thiolates. With respect to the Cys --> Ser substitution, the S covalency decreases. Calculations indicate that the stronger bonding interaction of the alkoxide with the Fe relative to that of thiolate increases the energy of the Fe d orbitals and reduces their bonding interaction with the remaining cysteines. The solvent effects support a surface solvent tuning contribution to electron transfer, and the Cys --> Ser result provides an explanation for the change in properties of related iron-sulfur sites with this mutation.

The challenges of determining metal-protein affinities.

A key property of metallo-proteins and -enzymes is the affinity of metal ion M for protein ligand P as defined by the dissociation constant KD = [M][P]/[MP]. Its accurate determination is essential for a quantitative understanding of metal selection and speciation. However, the surfaces of proteins are defined by the sidechains of amino acids and so abound in good metal ligands (e.g., imidazole of histidine,thiol of cysteine, carboxylate of aspartic and glutamic acids, etc.). Consequently, adventitious binding of metal ions to protein surfaces is common with KD values > or = 10(-6) M. On the other hand, transport proteins responsible for 'chaperoning' essential metals to their cellular destinations appear to bind the metal ions selectively (KD < 10(-7) M, both for speciation and to minimise the toxic effects of 'free' metal ions. These ions are normally bound with still higher affinities at their ultimate destinations (the active sites of metallo-proteins and -enzymes). This review surveys possible approaches to estimation of these dissociation constants and pinpoints the various problems associated with each approach.

A water-soluble bis(thiosemicarbazone) ligand. a sensitive probe and metal buffer for zinc.

The first member of a water-soluble family of bis(thiosemicarbazone) ligands is reported. It forms a 1:1 complex with Zn(II) that absorbs intensely in the visible region (lambda(max) = 414 nm; epsilon = 1.8(4) x 10(4) M(-1) cm(-1); pH 7.3). Its affinity for Zn(II) (K(D) = 5.9(3) x 10(-9) M at pH 7.3) was determined by competition with ligand ethylene glycol O,O'-bis(2-aminoethyl)-N,N,N',N'-tetraacetic acid. Its potential application as a chromophoric probe was demonstrated by estimation of the Zn(II) binding affinities of the soluble metal-binding domains of two plant metal-transporting proteins.

Metal binding affinities of Arabidopsis zinc and copper transporters: selectivities match the relative, but not the absolute, affinities of their amino-terminal domains.

HMA2, HMA4, and HMA7 are three of the eight heavy metal transporting P(1B)-type ATPases in the simple plant Arabidopsis thaliana. The first two transport Zn(2+), and the third transports Cu(+). Each protein contains soluble N-terminal metal-binding domains (MBDs) that are essential for metal transport. While the MBD of HMA7 features a CxxC sequence motif characteristic of Cu(I) binding sites, those of HMA2 and HMA4 contain a CCxxE motif, unique for plant Zn(2+)-ATPases. The three MBDs HMA2n (residues 1-79), HMA4n (residues 1-96), and HMA7n (residues 56-127) and an HMA7/4n chimera were expressed in Escherichia coli. The chimera features the ICCTSE motif from HMA4n inserted in place of the native MTCAAC motif of HMA7n. Binding affinities for Zn(II) and Cu(I) of each MBD were determined by ligand competition with a number of chromophoric probes. The challenges of using these probes reliably were evaluated, and the relative affinities of the MBDs were verified by independent cross-checks. The affinities of HMA2n and HMA4n for Zn(II) are higher than that of HMA7n by a factor of 20-30, but the relative affinities for Cu(I) are inverted by a factor of 30-50. These relativities are consistent with their respective roles in metal selection and transportation. Chimera HMA7/4n binds Cu(I) with an affinity between those of HMA4n and HMA7n but binds Zn(II) more weakly than either parent protein does. The four MBDs bind Cu(I) more strongly than Zn(II) by factors of >10(6). It is apparent that the individual MBDs are not able to overcome the large thermodynamic preference for Cu(+) over Zn(2+). This information highlights the potential toxicity of Cu(+) in vivo and why copper sensor proteins are approximately 6 orders of magnitude more sensitive than zinc sensor proteins. Metal speciation must be controlled by multiple factors, including thermodynamics (affinity), kinetics (including protein-protein interactions), and compartmentalization. The structure of Zn(II)-bound HMA4n defined by NMR confirmed the predicted ferredoxin betaalphabetabetaalphabeta fold. A single Zn atom was modeled onto a metal-binding site with protein ligands comprising the two thiolates and the carboxylate of the CCxxE motif. The observed (113)Cd chemical shift in [(113)Cd]HMA4n was consistent with a Cd(II)S(2)OX (X = O or N) coordination sphere. The Zn(II) form of the Cu(I) transporter HMA7n is a monomer in solution but crystallized as a polymeric chain [(Zn(II)-HMA7n)(m)]. Each Zn(II) ion occupied a distorted tetrahedral site formed from two Cys ligands of the CxxC motif of one HMA7n molecule and the amino N and carbonyl O atoms of the N-terminal methionine of another.

Unprecedented binding cooperativity between Cu(I) and Cu(II) in the copper resistance protein CopK from Cupriavidus metallidurans CH34: implications from structural studies by NMR spectroscopy and X-ray crystallography.

The bacterium Cupriavidus metallidurans CH34 is resistant to high environmental concentrations of many metal ions, including copper. This ability arises primarily from the presence of a large plasmid pMOL30 which includes a cluster of 19 cop genes that respond to copper. One of the protein products CopK is induced at high levels and is expressed to the periplasm as a small soluble protein (8.3 kDa). Apo-CopK associates in solution to form a dimer (K(D) approximately 10(-5) M) whose structure was defined by NMR and X-ray crystallography. The individual molecules feature two antiparallel beta-sheets arranged in a sandwich-like structure and interact through C-terminal beta-strands. It binds Cu(II) with low affinity (K(D)(Cu(II)) > 10(-6) M) but Cu(I) with high affinity (K(D)(Cu(I)) = 2 x 10(-11) M). Cu(I)-CopK was also a dimer in the solid state and featured a distorted tetrahedral site Cu(I)(S-Met)(3)(NCS). The isothiocyanato ligand originated from the crystallization solution. Binding of Cu(I) or Ag(I), but not of Cu(II), favored the monomeric form in solution. While Ag(I)-CopK was stable as isolated, Cu(I)-CopK was moderately air-sensitive due to a strong binding cooperativity between Cu(I) and Cu(II). This was documented by determination of the Cu(I) and Cu(II) binding affinities in the presence of the other ion: K(D)(Cu(I)) = 2 x 10(-13) M and K(D)(Cu(II)) = 3 x 10(-12) M, that is, binding of Cu(II) increased the affinity for Cu(I) by a factor of approximately 10(2) and binding of Cu(I) increased the affinity for Cu(II) by a factor of at least 10(6). Stable forms of both Cu(I)Cu(II)-CopK and Ag(I)Cu(II)-CopK were isolated readily. Consistent with this unprecedented copper binding chemistry, NMR spectroscopy detected three distinct forms: apo-CopK, Cu(I)-CopK and Cu(I)Cu(II)-CopK that do not exchange on the NMR time scale. This information provides a valuable guide to the role of CopK in copper resistance.

Interaction of cisplatin and analogues with a Met-rich protein site.

The chaperone protein CopC from Pseudomonas syringae features high-affinity binding sites (K (D) ~ 10(-13) M) for both Cu(I) (Met-rich) and Cu(II) (His-rich). When presented with these sites in the apoprotein, electrospray ionisation mass spectrometry confirmed that cis-Pt(NH(3))(2)Cl(2) (cisplatin) and the fragments [Pt(II)L](2+) (L is 1,2-diaminoethane, 2,2'-bipyridine) occupied the Cu(I) site specifically in the 1:1 Pt-CopC adducts (purified by cation-exchange chromatography). The cis-Pt(NH(3))(2) fragment was not present in these adducts (the dominant product for cisplatin was Pt-CopC in which all original ligands were displaced), while bidentate ligands L were retained in LPt-CopC adducts. In the context of the Met-rich Cu(I) pump Ctr1 as a significant entry point for cisplatin into mammalian cells, the present work confirms the ability of Met-rich sites in proteins to remove all ligands from cisplatin. It focuses attention on the potential of proteins that are part of the natural copper transport pathways to sequester the drug. These pathways are worthy of further study at the molecular level.

Gas-phase fragmentation of polyoxotungstate anions.

A series of phospho-polyoxotungstate anions was transferred to the gas phase via electrospray ionization (ESI), and the anions' fragmentation was examined by collision-induced dissociation (CID). The anions included [PW12O40]3-, [P2W18O62]6-, and {Co4(H2O)2][PW9O34]2}10- as well as lacunary and metal-substituted derivatives such as [PW11O39]7- and [MPW11O39]5- (M = Co(II), Ni(II), Cu(II)). Common species observed in the mass spectra arose from protonation and alkali metal cationization of the precursor ions. Additional species arising from the formal loss of oxide from the precursor species were also observed, presumably formed via protonation and the loss of an oxo ligand as water. These processes of protonation/cationization and the loss of water both led to species with reduced gas-phase anionic charges, and their formation appears to be driven by the enhanced effects of Coulombic repulsion in the desolvated species generated during transfer to the gas phase via ESI. Fragmentation of selected species was examined by multistage mass spectrometry experiments employing CID. Fragmentation occurred via multiple reaction channels, leading to pairs of complementary product anions whose total stoichiometry and charge matched those of the precursor anion. For example, [PW12O40]3- fragmented to give pairs of product ions of general formulas [W(x)O(3x+1)]2- and [PW(12-x)O(39-3x)]- (x = 6-9), with the most intense pair being [W6O19]2- and [PW6O21]-. Similar ions were also observed for fragmentation of [P2W18O61]4- (derived from the loss of water from [P2W18O62]6-). The lacunary and M(II)-substituted lacunary systems fragmented via related pathways, with the latter generating additional fragment ions due to the presence of M(II). These results highlight the usefulness of ESI-MS in the characterization of complex polyoxometalate anion clusters.

Molecular Mechanisms of Glutaredoxin Enzymes: Versatile Hubs for Thiol-Disulfide Exchange between Protein Thiols and Glutathione.

The tripeptide glutathione (GSH) and its oxidized form glutathione disulfide (GSSG) constitute a key redox couple in cells. In particular, they partner protein thiols in reversible thiol-disulfide exchange reactions that act as switches in cell signaling and redox homeostasis. Disruption of these processes may impair cellular redox signal transduction and induce redox misbalances that are linked directly to aging processes and to a range of pathological conditions including cancer, cardiovascular diseases and neurological disorders. Glutaredoxins are a class of GSH-dependent oxidoreductase enzymes that specifically catalyze reversible thiol-disulfide exchange reactions between protein thiols and the abundant thiol pool GSSG/GSH. They protect protein thiols from irreversible oxidation, regulate their activities under a variety of cellular conditions and are key players in cell signaling and redox homeostasis. On the other hand, they may also function as metal-binding proteins with a possible role in the cellular homeostasis and metabolism of essential metals copper and iron. However, the molecular basis and underlying mechanisms of glutaredoxin action remain elusive in many situations. This review focuses specifically on these aspects in the context of recent developments that illuminate some of these uncertainties.