Investigation of the structure of the blue copper protein from Rhus vernicifera stellacyanin by 1H nuclear magnetic resonance spectroscopy.

The 270-MHz 1H nuclear magnetic resonance spectra of Cu(II), Cu(I), and apo-stellacyanin are reported and compared. The data indicate that little conformational change occurs on reduction of the protein or on removing the copper ion. In the aromatic region of the spectra of the holoprotein, resonances associated with two freely titrating histidines are observed. Two additional sharp resonances are observed in the spectra of the apostellacyanin which are tentatively assigned to additional histidines. This result requires that not more than two histidines can be ligands since there are only four histidines in the whole protein. The absence of methionine has been reported and is one of the possible causes for the difference between stellacyanin and the other copper blue proteins. A comparison of these data with those available for other blue copper proteins, in conjunction with the sequence information, leads to a proposed structure for the copper site in stellacyanin.     

The role of copper binding in the conformation of stellacyanin

The binding of Cu²⁺ to apostellacyanin occurs in two steps. The first step consists of a fast equilibrium reaction involving binding of copper to the protein in a non-native, though specific way, as shown by electron paramagnetic resonance measurements. All the spectroscopic properties of native stellacyanin are recovered in a slower monomolecular process (k = 7.5 × 10^?3 sec^?1 at 25 °C) characterized by high activation energy (ΔH_a = 22 kcal mole^?1) and low activation entropy (ΔS_a = 3.0 cal deg^?1 mole^?1). The second step parallels a conformational change of the copper-bound protein molecule. A large difference of the tyrosyl residues pKs is found between holo- and apostellacyanin. In the latter the tyrosyl residues appear to be more exposed to solvent perturbations. Ammonia or monovalent anions such as N₃^?, SCN^?, and Cl^? have a catalytic effect on the second step of the reaction, roughly proportional to their first binding constant to aqueous copper. It is suggested that they may compete for a non-native bond of the copper to the protein, thus rendering the conformational change easier.The effect of Ag³ and Hg²⁺ on the recombination reaction with copper is discussed in terms of conformation of the metal-bound protein.     

Resonance Raman spectroscopy of amicyanin, a blue copper protein from Paracoccus denitrificans

The copper binding site of amicyanin from Paracoccus denitrificans has been examined by resonance Raman spectroscopy. The pattern of vibrational modes is clearly similar to those of the blue copper proteins azurin and plastocyanin. Intense resonance-enhanced peaks are observed at 377, 392, and 430 cm-1 as well as weaker overtones and combination bands in the high frequency region. Most of the peaks below 500 cm-1 shift 0.5-1.5 cm-1 to lower energy when the protein is exposed to D2O. Based on the pattern of conserved amino acids, the axial type EPR spectrum, and the resonance Raman spectrum, it is proposed that the copper binding site in amicyanin contains a Cu(II) ion in a distorted trigonal planar geometry with one cysteine and two histidine ligands and an axial methionine ligand at a considerably longer distance. Furthermore, the presence of multiple intense Raman peaks in the 400 cm-1 region which are sensitive to deuterium substitution leads to the conclusion that the Cu-S stretch is coupled with internal ligand vibrational modes and that the sulfur of the cysteine ligand is likely to be hydrogen-bonded to the polypeptide backbone.     

Modification of cysteine 111 in Cu/Zn superoxide dismutase results in altered spectroscopic and biophysical properties

Cu/Zn superoxide dismutase (SOD) mutations are involved in about 20% of all cases of familial amyotrophic lateral sclerosis (FALS). Recently, it has been proposed that aberrant copper activity may be occurring within SOD at an alternative binding, and cysteine 111 has been identified as a potential copper ligand. Using a commercial source of human SOD isolated from erythrocytes, an anomalous absorbance at 325 nm was identified. This unusual property, which does not compromise SOD activity, had previously been shown to be consistent with a sulfhydryl modification at a cysteine residue. Here, we utilized limited trypsin proteolysis and mass spectrometry to show that the modification has a mass of 32 daltons and is located at cysteine 111. The reaction of SOD with sodium sulfide, which can react with cysteine to form a persulfide group, and with potassium cyanide, which can selectively remove persulfide bonds, confirmed the addition of a persulfide group at cysteine 111. Gel electrophoresis and glutaraldehyde cross-linking revealed that this modification makes the acid-induced denaturation of SOD fully irreversible. Furthermore, the modified protein exhibits a slower acid-induced unfolding, and is more resistant to oxidation-induced aggregation caused by copper and hydrogen peroxide. Thus, these results suggest that cysteine 111 can have a biochemical and biophysical impact on SOD, and suggest that it can interact with copper, potentially mediating the copper-induced oxidative damage of SOD. It will be of interest to study the role of cysteine 111 in the oxidative damage and aggregation of toxic SOD mutants.     

High affinity binding between copper and full-length prion protein identified by two different techniques

The cellular prion protein is known to be a copper-binding protein. Despite the wide range of studies on the copper binding of PrP, there have been no studies to determine the affinity of the protein on both full-length prion protein and under physiological conditions. We have used two techniques, isothermal titration calorimetry and competitive metal capture analysis, to determine the affinity of copper for wild type mouse PrP and a series of mutants. High affinity copper binding by wild type PrP has been confirmed by the independent techniques indicating the presence of specific tight copper binding sites up to femtomolar affinity. Altogether, four high affinity binding sites of between femto- and nanomolar affinities are located within the octameric repeat region of the protein at physiological pH. A fifth copper binding site of lower affinity than those of the octameric repeat region has been detected in full-length protein. Binding to this site is modulated by the histidine at residue 111. Removal of the octameric repeats leads to the enhancement of affinity of this fifth site and a second binding site outside of the repeat region undetected in the wild type protein. High affinity copper binding allows PrP to compete effectively for copper in the extracellular milieu. The copper binding affinities of PrP have been compared with those of proteins of known function and are of magnitudes compatible with an extracellular copper buffer or an enzymatic function such as superoxide dismutase like activity.     

Acetate C-C bond formation and decomposition in the anaerobic world: the structure of a central enzyme and its key active-site metal cluster

The structure of carbon monoxide dehydrogenase/acetyl-coenzyme A synthase (CODH/ACS), a central enzyme in the anaerobic metabolism of acetyl-coenzyme A (acetyl-CoA), has been solved to a resolution of 2.2A. The active-site metal cluster responsible for catalyzing acetyl C-C bond synthesis and cleavage, designated the A center, was identified as an Fe(4)S(4) iron sulfur cluster with one of its cysteine thiolates acting as a bridge to an adjacent binuclear metal site. Nickel was found at one position in the binuclear site and the other metal was indicated to be copper - a surprising result, implying a previously unrecognized role for copper. Details of the A center provided new insight into the unusual organometallic mechanism of acetyl C-C bond formation and cleavage, with substantial conformational changes indicated for binding of the large methylcorrinoid protein substrate, and a unique intramolecular channel acting to contain carbon monoxide within the protein and transfer it to the site needed for acetyl-CoA synthesis.     

Periplasmic disulfide isomerase DsbC is involved in the reduction of copper binding protein CueP from Salmonella enterica serovar Typhimurium

Salmonella enterica serovar Typhimurium (S. Typhimurium) is a facultative intracellular pathogen with the ability to survive and replicate in macrophages. Periplasmic copper binding protein CueP is known to confer copper resistance to S. Typhimurium, and has been implicated in ROS scavenge activity by transferring the copper ion to a periplasmic superoxide dismutase or by directly reducing the copper ion. Structural and biochemical studies on CueP showed that its copper binding site is surrounded by conserved cysteine residues. Here, we present evidence that periplasmic disulfide isomerase DsbC plays a key role in maintaining CueP protein in the reduced state. We observed purified DsbC protein efficiently reduced the oxidized form of CueP, and that it acted on two (Cys104 and Cys172) of the three conserved cysteine residues. Furthermore, we found that a surface-exposed conserved phenylalanine residue in CueP was important for this process, which suggests that DsbC specifically recognizes the residue of CueP. An experiment using an Escherichia coli system confirmed the critical role played by DsbC in the ROS scavenge activity of CueP. Taken together, we propose a molecular insight into how CueP collaborates with the periplasmic disulfide reduction system in the pathogenesis of the bacteria.     

Contribution of individual histidines to prion protein copper binding

The prion protein is well-established as a copper binding protein. The N-terminus of the protein contains an octameric repeat region with each of the four repeats containing a histidine. The N-terminus has two additional histidines distal to the repeat region that has been commonly known as the fifth site. While binding of copper by the protein has been extensively studied, the contribution of each histidine to copper binding in the full-length protein has not. Here we used a battery of mutants of the recombinant mouse prion protein to assess copper binding with both isothermal titration calorimetry and cyclic voltammetry. The findings indicate that there is extensive cooperativity between different binding sites in the protein. The two highest-affinity binding events occur at the fifth site and at the octameric repeat region. However, the first binding is that to the octameric repeat region. Subsequent binding events after the two initial binding events have lower affinities within the octameric repeat region.     

The homodimeric hemoglobin from Scapharca can be locked into new cooperative structures upon reaction of Cys92, located at the subunit interface, with organomercurials.

In the cooperative, homodimeric hemoglobin from Scapharca inaequivalvis, HbI, the subunit interface is formed by the heme-carrying E and F helices and contains the only cysteine residue of the globin chain (Cys92, F2) in an area which changes from hydrophilic to hydrophobic upon oxygenation. Binding of organomercurials to HbI is cooperative and entails major quaternary rearrangements. The reaction of Cys92 with p-chloromercuri-benzoate (PMB) and p-nitro-o-chloromercuriphenol (PN), a sensitive reporter of the cysteine microenvironment at neutral pH values, has been followed in stopped flow experiments. Kinetic evidence for the cooperativity of mercurial binding has been obtained and the rate of the corresponding conformational transition has been estimated. As expected PN, but not PMB, is able to monitor the oxygen-linked change of the cysteine microenvironment. The modification of Cys92 with PN has unique functional effects. In PN-reacted HbI cooperativity is maintained, albeit to a different extent, depending on the ligation state of the protein during mercaptide formation. It may be envisaged that PN locks the protein into new, cooperative, quaternary structures stabilized by hydrogen bonding interactions between the ionized nitrophenol moiety and the contralateral subunit.     

Reactivity of ligand-swapped mutants of the SCO protein from Bacillus subtilis. Isomers of the CCH metal binding motif

The Synthesis of Cytochrome Oxidase protein, or SCO protein, is required for the assembly of cytochrome c oxidase in many mitochondrial and bacterial respiratory chains. SCOs have been proposed to deliver copper to the Cu_A site of cytochrome c oxidase. We have reported that Bacillus subtilis SCO (i.e., BsSCO) binds Cu(II) with high-affinity via a two-step process mediated by three conserved residues (i.e., two cysteines and one histidine, or the CCH motif). A remarkable feature in the reaction of reduced (i.e., di-thiol) BsSCO with copper is that it does not generate any of the disulfide form of BsSCO. This molecular aversion is proposed to be a consequence of a binding mechanism in which the initial copper complex of BsSCO does not involve cysteine, but instead involves nitrogen ligands. We test this proposal here by constructing two isomers of BsSCO in which the conserved copper binding residues (i.e., the CCH-motif) are retained, but their positions are altered. In these variants the two cysteines are exchanged with histidine, and both react transiently with copper (II) with distinct kinetic profiles. The reaction generates Cu(I) and the protein is oxidized to its disulfide form. EPR analysis supports a copper binding model in which cysteine, which is at the “histidine position” in the mutant, is part of an initial encounter complex with copper. When cysteine is the initial ligating residue an oxidation reaction ensues. In contrast initial binding to native BsSCO uses nitrogen-based ligands, and thereby avoids the opportunity for thiol oxidation.     

Conformational stabilization at the active site of molluskan (Rapana thomasiana) hemocyanin by a cysteine-histidine thioether bridge A study by mass spectrometry and molecular modeling

In some type-3 copper proteins (molluskan hemocyanin, catechol oxidase and fungal tyrosinase) one of the histidine residues, liganding the Cu(A) atom of the dinuclear copper active site, is covalently linked to a cysteine residue by a thioether bridge. The purpose of this study was to disclose the function of this bridge. Mass spectral analysis of a peptide, isolated from Rapana thomasiana (gastropodan mollusk) hemocyanin, indicated a stabilization of the peptide structure in the region of the bridge. Molecular modeling of three thioether containing type-3 copper proteins using the dead-end elimination method showed that the concerned histidine would be very flexible if not linked to the cysteine. Also, the side chain orientation of the histidine is rather exceptional, as evidenced by statistical data from the protein databank. It is suggested that the role of the bridge is to fix the histidine in an orientation that is optimal for coordination of the Cu(A) atom.     

A conserved cis-proline precludes metal binding by the active site thiolates in members of the thioredoxin family of proteins

Many thioredoxin-fold proteins possess a conserved cis-proline located in their C-terminal portions. This residue, as well as catalytic and resolving cysteines, is a key functional group in the active sites of these thiol-disulfide oxidoreductases. However, the specific function of the proline is poorly understood, and some thioredoxin-fold proteins lack this residue. Herein, we found that mutation of a cis-proline, Pro75, in human thioredoxin to serine, threonine, or alanine leads to the formation of an Fe2-S2 cluster in this protein. Further mutagenesis studies revealed that the first cysteine in the CxxC motif and a cysteine in the C-terminal region of the protein were responsible for metal binding. Replacement of Pro75 with arginine, a residue that occurs in place of Pro in peroxiredoxins, also led to the formation of the cluster in the thioredoxin. In addition, we found that mutation of the TxxC active site in a peroxiredoxin to the CxxC form could lead to coordination of an Fe2-S2 cluster in these proteins in vitro. Sco1, a distantly related thioredoxin-fold protein, has histidine in place of the cis-proline, and this residue binds copper. The Pro75His mutation led to increased copper binding by human thioredoxin when cells were grown in the presence of this trace element. Taken together, our data suggest that an important function of Pro75 in human thioredoxin, and likely other members of this superfamily, is to prevent metal binding by the reactive thiolate-based active site.     

Copper-induced conformational changes in the N-terminal domain of the Wilson disease copper-transporting ATPase

The Wilson disease copper-transporting ATPase plays a critical role in the intracellular trafficking of copper. Mutations in this protein lead to the accumulation of a toxic level of copper in the liver, kidney, and brain followed by extensive tissue damage and death. The ATPase has a novel amino-terminal domain ( approximately 70 kDa) which contains six repeats of the copper binding motif GMTCXXC. We have expressed and characterized this domain with respect to the copper binding sites and the conformational consequences of copper binding. A detailed analysis of this domain by X-ray absorption spectroscopy (XAS) has revealed that each binding site ligates copper in the +1 oxidation state using two cysteine side chains with distorted linear geometry. Analysis of copper-induced conformational changes in the amino-terminal domain indicates that both secondary and tertiary structure changes take place upon copper binding. These copper-induced conformational changes could play an important role in the function and regulation of the ATPase in vivo. In addition to providing important insights on copper binding to the protein, these results suggest a possible mechanism of copper trafficking by the Wilson disease ATPase.     

Crystal structure of a sulfur carrier protein complex found in the cysteine biosynthetic pathway of Mycobacterium tuberculosis

The structure of the protein complex CysM-CysO from a new cysteine biosynthetic pathway found in the H37Rv strain of Mycobacterium tuberculosis has been determined at 1.53 A resolution. CysM (Rv1336) is a PLP-containing beta-replacement enzyme and CysO (Rv1335) is a sulfur carrier protein with a ubiquitin-like fold. CysM catalyzes the replacement of the acetyl group of O-acetylserine by CysO thiocarboxylate to generate a protein-bound cysteine that is released in a subsequent proteolysis reaction. The protein complex in the crystal structure is asymmetric with one CysO protomer binding to one end of a CysM dimer. Additionally, the structures of CysM and CysO were determined individually at 2.8 and 2.7 A resolution, respectively. Sequence alignments with homologues and structural comparisons with CysK, a cysteine synthase that does not utilize a sulfur carrier protein, revealed high conservation of active site residues; however, residues in CysM responsible for CysO binding are not conserved. Comparison of the CysM-CysO binding interface with other sulfur carrier protein complexes revealed a similarity in secondary structural elements that contribute to complex formation in the ThiF-ThiS and MoeB-MoaD systems, despite major differences in overall folds. Comparison of CysM with and without bound CysO revealed conformational changes associated with CysO binding.     

Interactions between metals and alpha-synuclein--function or artefact?

alpha-synuclein is one of a family of proteins whose function remains unknown. This protein has become linked to a number of neurodegenerative disease although its potential causative role in these diseases remains mysterious. In diseases such as Parkinson's disease and Lewy body dementias, alpha-synuclein becomes deposited in aggregates termed Lewy bodies. Also, some inherited forms of Parkinson's diseases are linked to mutations in the gene for alpha-synuclein. Studies have mostly focussed on what causes the aggregation of the protein but, like many amyloidogenic proteins associated with a neurodegenerative disorder, this protein has now been suggested to bind copper. This finding is currently controversial. This review examines the evidence that alpha-synuclein is a copper binding protein and discusses whether this has any significance in determining the function of the protein or whether copper binding is at all necessary for aggregation.     

Domains I and III of the human copper chaperone for superoxide dismutase interact via a cysteine-bridged Dicopper(I) cluster

Copper binding to the human copper chaperone for superoxide dismutase (hCCS) has been investigated by X-ray absorption spectroscopy. Stoichiometry measurements on the dialyzed, as-isolated protein indicated that up to 3.5 Cu ions bound per hCCS molecule. Reduction with either sodium dithionite or dithiothreitol decreased the copper binding ratio to 2 coppers per hCCS monomer. Analysis of the as-isolated EXAFS data indicated coordination of Cu by a mixture of S and N backscatterers, suggestive of heterogeneous binding of copper between Cu-cysteine binding sites of domain I or III and copper-histidine SOD1-like metal binding sites of domain II. The best fit was obtained with 1.6 Cu-S (cysteine) at 2.24 A (2sigma(2) = 0.011 A(2)) and 1.1 N (histidine) at 1.98 A (2sigma(2) = 0.005 A(2)). A peak of variable intensity in the Fourier transform (FT) of the as-isolated protein at 2.7 A was suggestive of the presence of a heavy atom scatterer such as Cu. Analysis of the dithionite- and DTT-reduced derivatives indicated that copper was lost from the histidine coordinating sites, resulting in a S-only environment with copper coordinated to three S backscatterers at 2. 26 A. The heavy atom scatterer peak was now prominent in the FT and could be well fit by a Cu-Cu interaction at 2.72 A. The data were best interpreted by a dinuclear mu(2)()-bridged cluster with doubly bridging cysteine ligands similar to the cluster proposed to exist in the cytochrome c oxidase chaperone COX17. Analysis of primary sequence and X-ray structural information on yeast CCS strongly suggests that this cluster bridges between domains I and III in hCCS. A mechanism for copper translocation is briefly discussed.     

Mapping and analysis of protein sulfhydryl groups by modification with 2-bromoacetamido-4-nitrophenol

A method for identifying cysteine-containing peptides in proteins is presented using 2-bromoacetamido-4-nitrophenol (BNP) to introduce an easily detectable probe. The formation of a covalent bond between the protein sulfhydryl group and the acetamido moiety of BNP introduces a chromophore with an absorbance maximum at 410 nm. The modified protein can then be cleaved with appropriate proteases and the resulting peptides separated by chromatographic methods. Monitoring the effluent at a single wavelength (405 nm) provides a rapid and simple method of detecting and isolating only those peptides which contain cysteine residue(s). The nitrophenol derivative is stable under conditions required for protease cleavage. The reagent is therefore useful for locating cysteine-containing peptides in protein digests and can be used to explore the accessibility of different cysteines under a variety of conditions. The ease of modification, specificity of reaction, product stability, and simple detection of modified peptides make BNP ideal for investigation of cysteine residues.     

Cobalt(2+) binding to human and tomato copper chaperone for superoxide dismutase: implications for the metal ion transfer mechanism

The copper chaperone for superoxide dismutase (CCS) gene encodes a protein that is believed to deliver copper ions specifically to copper-zinc superoxide dismutase (CuZnSOD). CCS proteins from different organisms share high sequence homology and consist of three distinct domains; a CuZnSOD-like central domain 2 flanked by domains 1 and 3, which contain putative metal-binding motifs. We report deduced protein sequences from tomato and Arabidopsis, the first functional homologues of CCS identified in plants. We have purified recombinant human (hCCS) and tomato (tCCS) copper chaperone proteins, as well as a truncated version of tCCS containing only domains 2 and 3. Their cobalt(2+) binding properties in the presence and absence of mercury(2+) were characterized by UV-vis and circular dichroism spectroscopies and it was shown that hCCS has the ability to bind two spectroscopically distinct cobalt ions whereas tCCS binds only one. The cobalt binding site that is common to both hCCS and tCCS displayed spectroscopic characteristics of cobalt(2+) bound to four or three cysteine ligands. There are only four cysteine residues in tCCS, two in domain 1 and two in domain 3; all four are conserved in other CCS sequences including hCCS. Thus, an interaction between domain 1 and domain 3 is concluded, and it may be important in the copper chaperone mechanism of these proteins.     

Manganese binding to the prion protein

There is considerable evidence that the prion protein binds copper. However, there have also been suggestions that prion protein (PrP) binds manganese. We used isothermal titration calorimetry to identify the manganese binding sites in wild-type mouse PrP. The protein showed two manganese binding sites with affinities that would bind manganese at concentrations of 63 and 200 mum at pH 5.5. This indicates that PrP binds manganese with affinity similar to other known manganese-binding proteins. Further study indicated that the main manganese binding site is associated with His-95 in the so-called "fifth site" normally associated with copper binding. Additionally, it was shown that occupancy by copper does not prevent manganese binding. Under these conditions, manganese binding resulted in an altered conformation of PrP, displacement of copper, and altered redox chemistry of the metal-protein complex. Cyclic voltammetric measurements suggested a complex redox chemistry involving manganese bound to PrP, whereas copper-bound PrP was able to undergo fully reversible electron cycling. Additionally, manganese binding to PrP converted it to a form able to catalyze aggregation of metal-free PrP. These results further support the notion that manganese binding could cause a conformation change in PrP and trigger changes in the protein similar to those associated with prion disease.     

The cooperative binding of chromosomal protein HMG-14 to nucleosome cores is reduced by single point mutations in the nucleosomal binding domain.

Mutants of human chromosomal protein HMG-14 were generated by site directed mutagenesis and used to study functional domains in this protein. A replacement of serine by cysteine at position 7 did not affect the binding of the protein to nucleosome cores. The sulfhydryl group in the nucleosome-bound protein is accessible to modifying agents suggesting that position 7 in the protein is not in close contact with either the DNA or the histones in the core particles. Under cooperative binding conditions, replacements of alanine by proline at position 21, or of lysine by cysteine at position 26, decreased the affinity of the protein for nucleosome cores 6.7- and 3-fold respectively. In contrast, the non-cooperative mode of binding was only minimally affected. A replacement of glutamic acid by glutamine at position 76 caused only minor changes in the binding of the protein to the cores. The results indicate that single point mutations, which change either the conformation or change in the nucleosomal binding domain of the protein, significantly reduce the ability of the HMG-14 protein to bind to nucleosome cores. We suggest that in chromatin the protein binds to nucleosomes in a cooperative manner and that upon binding to nucleosomes the protein acquires a distinct conformation.