Thermodynamics of the acid transition in blue copper proteins

The thermodynamic parameters of the conformational transition occurring at low pH (acid transition, AT) in blue copper proteins, involving protonation and detachment from the Cu(I) ion of one histidine ligand, have been determined electrochemically for spinach and cucumber plastocyanins, Rhus vernicifera stellacyanin, cucumber basic protein (CBP), and Paracoccus versutus amicyanin. These data were obtained from direct protein electrochemistry experiments carried out at varying pH and temperature. For all species but CBP, the overall conformational change turns out to be exothermic. The entropy change is remarkably species-dependent. In particular, we found that (i) the balance of bond breaking/formation favors the acid transition in plastocyanins, which show remarkably negative DeltaH degrees '(AT) values, and (ii) the transition enthalpy turns out to be much less negative (or even positive) for the two phytocyanins (stellacyanin and CBP): for these species, the transition turns out to be observable thanks to the favorable (positive) entropy change. Thus, it is apparent that the thermodynamic "driving force" for this transition is enthalpic for the plastocyanins and entropic for the phytocyanins. Amicyanin is an intermediate case in which both enthalpic and entropic terms favor the transition. Under the assumption that the transition entropy originates from solvent reorganization effects, which are known to involve compensative enthalpy and entropy changes, the free energy change of the transition would also correspond to the enthalpy change due to bond breaking/formation in the first coordination sphere of the metal and in its immediate environment. Indeed, this term turns out to be very similar for the proteins investigated, in line with the conservation of the Cu(I)-His bond strengths in these species, except for amicyanin, for which the greater exothermicity of the transition can be ascribed to peculiar features of the active site.     

On the role of strain in blue copper proteins

Theoretical investigations of the structure and function of the blue copper proteins are described. We have studied the optimum vacuum geometry of oxidised and reduced copper sites, the relative stability of trigonal and tetragonal Cu(II) structures, the relation between the structure and electronic spectra, the reorganisation energy, and reduction potentials. Our calculations give no support to the suggestion that strain plays a significant role in the function of these proteins; on the contrary, our results show that the structures encountered in the proteins are close to their optimal vacuum geometries (within 7 kJ/mol). We stress the importance of defining what is meant by strain and of quantifying strain energies or forces in order to make strain hypotheses testable.     

Functionally accepted insertions of proteins within protein domains

Experiments were designed to explore the tolerance of protein structure and folding to very large insertions of folded protein within a structural domain. Dihydrofolate reductase and beta-lactamase have been inserted in four different positions of phosphoglycerate kinase. The resultant chimeric proteins are all overexpressed, and the host as well as the inserted partners are functional. Although not explicitly designed, functional coupling between the two fused partners was observed in some of the chimeras. These results show that the tolerance of protein structures to very large structured insertions is more general than previously expected and supports the idea that the natural sequence continuity of a structural domain is not required for the folding process. These results directly suggest a new experimental approach to screen, for example, for folded protein in randomized polypeptide sequences.     

Bacterial binding protein-dependent permeases: characterization of distinctive signatures for functionally related integral cytoplasmic membrane proteins

Bacterial binding protein-dependent transport systems belong to the superfamily of ABC transporters, which is widely distributed among living organisms. Their hydrophobic membrane proteins are the least characterized components. The primary structures of 61 integral membrane proteins from 35 uptake systems were compared in order to characterize a short conserved hydrophilic segment, with a consensus EAA … G ………-I - LP, located approximately 100 residues from the C-terminus. Secondary structure predictions indicated that this conserved region might be formed by two amphipathic α-helices connected by a loop containing the invariant G residue. We classified the conserved motifs and found that membrane proteins from systems transporting structurally related substrates specifically display a greater number of identical residues in the conserved region. We determined a consensus for each class of membrane protein and showed that these can be considered as signatures.     

Increased uniformity in the response of the coomassie blue G protein assay to different proteins

Coomassie blue G dye-based protein assays are exceptionally convenient because of their simplicity, sensitivity, speed, and resistance to interfering chemicals, notably reducing agents and most buffers. A major problem with the assay is the variation in response to different proteins. The addition of NaOH to the protein assay reagent reduced the variation in the response of this assay to different proteins. In addition, the sensitivity of the assay is increased. The NaOH can be added either in a separate step to solubilize cells or membranes or directly to the reagent. Linear standard curves were obtained when the log of the absorbance was plotted against the log of the protein quantity.     

The prediction of reverse turns in the blue copper proteins and their copper cores

The frequencies of occurrence of the side chains in proteins in the first, second, third, and fourth positions of a reverse turn in a set of 26 nonredundant protein chains are shown in a table that lists cysteine and cystine side chains separately. This table was used to predict the reverse turns in poplar plastocyanin whose crystal structure is known (75% of the turn residues are correctly predicted but the overall accuracy of the predictions is only 66% in a turn-not-turn two-state model), and in three blue copper proteins whose crystal structures are being determined (cucumber plastocyanin and cucumber basic protein) or contemplated (Rhus vernificera stellacyanin). The copper cores proposed for cucumber basic protein and stellacyanin are discussed.     

Functionally different GPI proteins are organized in different domains on the neuronal surface

We have investigated the organization, on the plasma membrane and in detergent-insoluble membrane vesicles, of two neuronal glycosylphosphatidylinositol-anchored (GPI) proteins: Thy-1, a negative regulator of transmembrane signalling; and prion protein, whose rapid endocytosis and Cu(2+) binding suggest that it functions in metal ion uptake. Prion protein occurred on the neuronal surface at high density in domains, located primarily at the cell body, which were relatively soluble in detergent. Thy-1, although much more abundantly expressed on neurons, occurred at lower density over much of the surface of neurites (and in lower abundance at the cell body) in domains that were highly resistant to detergent solubilization. Detergent-insoluble membrane vesicles contained Thy-1 at a density similar to that on the neuronal surface. Vesicles containing each protein could be separated by immunoaffinity isolation; lectin binding showed that they were enriched in different glycoproteins. Our results demonstrate a structural diversity of the domains occupied by functionally different GPI proteins.     

Interpretation of the temperature-dependent color of blue copper protein mutants

The electronic absorption spectrum of the mutant of the blue copper protein amicyanin with a pseudoazurin loop (AmiPse) shows a remarkable temperature dependence. The absorption band at approximately 460 nm increases at low temperature while the transition at approximately 600 nm is not much affected by a variation of the temperature. An approximate density functional theory (DFT) study of the active site model [Cu(II)(imidazole)(2)(SCH(3))(S(CH(3))(2))](+) (protein backbone and solvation neglected) leads to two local minimum structures (axial and rhomb) which both have a geometry close to that typical for blue copper proteins. One (rhomb) has two structurally different histidine donors, and this geometry is also found in most experimental type 1 structures. The two forms axial and rhomb are distortional isomers and are energetically almost degenerate. The temperature dependence of the spectrum of AmiPse is interpreted with a temperature-dependent change of the relative population of the two local minimum structures with slightly different energy. The 460 nm transition is believed to be due to preferential population of the structure rhomb; this is in agreement with the published assignment of the high energy transition, based on thorough spectroscopic and computational studies. Consequences of a perturbation of the "gas phase" structures axial and rhomb by the protein and solvation are also discussed on the basis of published, experimentally observed structures and spectroscopic data.     

The blue copper protein gene of Alcaligenes faecalis S-6 directs secretion of blue copper protein from Escherichia coli cells.

The gene encoding a blue copper protein (a member of the pseudoazurins) of 123 amino acid residues, containing a single type I Cu2+ ion, was cloned from Alcaligenes faecalis S-6. The nucleotide sequence of the coding region, as well as the 5'- and 3'-flanking regions, was determined. The deduced amino acid sequence after Glu-24 coincided with the reported sequence of the blue protein, and its NH2-terminal sequence of 23 residues resembled a typical signal peptide. The cloned gene was expressed under the control of the tac promoter in Escherichia coli, and the correctly processed blue protein was secreted into the periplasm. The blue protein produced in E. coli possessed the activity to transfer electrons to the copper-containing nitrite reductase of A. faecalis S-6 in vitro.     

Cu(I) analysis of blue copper proteins.

A simple colorimetric test for the Cu(I) content in blue copper proteins is described. The procedure is based on the formation of a complex between Cu(I) and 2,2'-biquinoline in an acetic acid medium. Analyses of spinach plastocyanin, Pseudomonas aeruginosa azurin and Rhus vernicifera stellacyanin show that the cysteine residue in the type 1 site does not induce Cu(II) reduction under our conditions. There is evidence in laccase samples for the presence of an endogenous reductant that can reduce 0.14 +/- 0.04 mol of Cu(II)/mol of protein; however, the addition of EDTA eliminates the interference. The analysis shows that 25 +/- 2% of the type 3 copper ions are in the reduced form in the resting enzyme and that 80 +/- 15% of the type 3 copper ions are reduced in preparations of type-2-depleted laccase. There is growing interest in the development of chemically modified forms of laccase, and our method should be very useful for establishing the valence state of the metal centres in the various derivatives.     

An outer-sphere hydrogen-bond network constrains copper coordination in blue proteins

In azurins and other blue copper proteins with relatively low reduction potentials (E(0) [Cu(II)/Cu(I)]<400 mV vs. normal hydrogen electrode), the folded polypeptide framework constrains both copper(II) and copper(I) in such a way as to tune the reduction potentials to values that differ greatly from those for most copper complexes. Largely conserved networks of hydrogen bonds organize and lock the rest of the folded protein structure to a loop that contains three of the ligands to copper. Changes in hydrogen bonds that allow copper(I) to revert more closely to its preferred geometry [relative to the copper(II) geometry] accordingly lead to an increase in E(0). This paper reports mutations in the ligand loop of amicyanin from P. denitrificans that relax the constraints on ligation for copper(I) and significantly raise E(0) for these mutants (for example 415+/-4 mV) relative to that of the native amicyanin (265+/-4 mV). These mutations also shift the pK(a) of a ligand histidine to below 5 relative to 7.0 in the wild type.     

The influence of axial ligands on the reduction potential of blue copper proteins

 The reduction potentials of blue copper sites vary between 180 and about 1000 mV. It has been suggested that the reason for this variation is that the proteins constrain the distance between the copper ion and its axial ligands to different values. We have tested this suggestion by performing density functional B3LYP calculations on realistic models of the blue copper proteins, including solvent effects by the polarizable continuum method. Constraining the Cu-S_Met bond length to values between 245 and 310 pm (the range encountered in crystal structures) change the reduction potential by less than 70 mV. Similarly, we have studied five typical blue copper proteins spanning the whole range of reduction potentials: stellacyanin, plastocyanin, azurin, rusticyanin, and ceruloplasmin. These studies included the methionine (or glutamine) ligand as well as the back-bone carbonyl oxygen group that is a ligand in azurin and is found at larger distances in the other proteins. The active-site models of these proteins show a variation in the reduction potential of about 140 mV, i.e., only a minor part of the range observed experimentally (800 mV). Consequently, we can conclude that the axial ligands have a small influence on the reduction potentials of the blue copper proteins. Instead, the large variation in the reduction potentials seems to arise mainly from variations in the solvent accessibility of the copper site and in the orientation of protein dipoles around the copper site. Received: 7 April 1999 / Accepted: 26 July 1999     

Modelling the impact of geometric parameters on the redox potential of blue copper proteins

The synthesis and structure of a homologous series of cationic N(2)S(2) copper(I) Schiff base complexes constructed using o-tert-butylthiobenzaldehyde and a series of terminal diamines (ethane, propane, butane) are reported. The complexes differ only in the length of the methylene chain between the imine groups. This simple modification forces the copper centre to shift geometry from a planar (1,2-diaminoethane) to a more distorted tetrahedral motif (1,4-diaminobutane). The redox potentials of the three cations were measured using cyclic voltammetry in donor (acetonitrile) and non-donor solvents (dichloromethane). The S-Cu-N angles for each complex are correlated against the respective redox potential allowing an analysis of the geometric impact on the redox potential in soft copper centres. The redox potential is observed to increase as the metal centre moves from a planar towards a tetrahedral motif. Comparing this data with the reported structures of the blue copper proteins (rusticyanin and plastocyanin) allows an assessment of the contribution of the geometry of the metal binding site to the operating potential of these proteins to be made.     

Spectrochemical studies on the blue copper protein azurin from Alcaligenes denitrificans

Spectroscopic and electrochemical studies, incorporating electronic spectra, electron paramagnetic resonance (EPR) spectra, resonance Raman (RR) spectra, and measurements of the redox potential, have been carried out on the blue copper protein azurin, from Alcaligenes denitrificans. These data are correlated with the refined crystal structure of this azurin and with corresponding data for other blue copper proteins. The electronic spectrum, characterized by an intense (epsilon = 5100 M-1 cm-1) charge-transfer band at 619 nm, the EPR spectral parameters (g perpendicular = 2.059, g parallel of = 2.255, A parallel of = 60 X 10(-4) cm-1), and the resonance Raman spectrum are similar to those obtained from other azurins and from plastocyanins. Both the electronic spectrum and the EPR spectrum are unchanged over the pH range 4-10.5, but major changes occur above pH 12 and below pH 3.5. A small reversible change occurs at pH approximately 11.4. In the RR spectrum the Cu-S stretching mode is shown to contribute to all of the five principal RR peaks. Deuterium substitution produces shifts in at least seven of the peaks; these shifts may be attributable, at least in part, to the NH...S hydrogen bond to the copper-ligated Cys-112. Measurements of the redox potential, using spectroelectrochemical methods, over the temperature range 4.8-40.0 degrees C, give values for delta H0' and delta S0' of -55.6 kJ mol-1 and -97.0 J K-1 mol-1, respectively. The redox potential of A. denitrificans azurin at pH 7.0, Eo', is 276 mV. These data are interpreted in terms of a copper site, in azurin, comprising three strong bonds, in an approximately trigonal plane, from Cys-112, His-46, and His-117 and much longer axial approaches from Met-121 and the peptide carbonyl oxygen of Gly-45. Spectral differences within the azurin family and between azurin and plastocyanin are attributed to differences in the strengths of these axial interactions. Likewise, the distinctly lower Eo values for azurins, as compared with plastocyanins, are related to the more copper(II)-like site in azurin [with a weaker Cu-S(Met) interaction and a Cu-O interaction not found in plastocyanin]. On the other hand, the relative constancy of the EPR parameters between azurin and plastocyanin suggests they are not strongly influenced by weakly interacting axial groups.     

Dimethoxyphenol oxidase activity of different microbial blue multicopper proteins

2,6-Dimethoxyphenol is a versatile substrate for Pyricularia oryzae laccase, PpoA from Marinomonas mediterranea, phenoxazinone synthase from Streptomyces antibioticus and mammalian ceruloplasmin. In addition, in cellular extracts of microorganisms expressing other blue multicopper proteins with no enzymatic activity previously described, such as Escherichia coli (copper resistance CueO), Pseudomonas syringae and Xanthomonas campestris (copper resistance CopA), Bacillus subtilis (sporulation protein CotA) and Saccharomyces cerevisiae (iron transporter Fet3p), laccase activity is detected under appropriate conditions. This oxidase activity can be spectrophotometrically followed by the oxidation of 2,6-dimethoxyphenol. Specific staining after SDS-PAGE is also possible for some of these proteins. This detection assay can facilitate the study of the multiple functions that such proteins seem to carry out in a variety of microorganisms.