Methionine-121 coordination determines metal specificity in unfolded<Emphasis Type="Italic"> Pseudomonas aeruginosa</Emphasis> azurin

Pseudomonas aeruginosa azurin binds copper so tightly that it remains bound even upon polypeptide unfolding. Copper can be substituted with zinc without change in protein structure, and also in this complex the metal remains bound upon protein unfolding. Previous work has shown that native-state copper ligands Cys112 and His117 are two of at least three metal ligands in the unfolded state. In this study we use isothermal titration calorimetry and spectroscopic methods to test if the native-state ligand Met121 remains a metal ligand upon unfolding. From studies on a point-mutated version of azurin (Met121Ala) and a set of model peptides spanning the copper-binding C-terminal part (including Cys112, His117 and Met121), we conclude that Met121 is a metal ligand in unfolded copper-azurin but not in the case of unfolded zinc-azurin. Combination of unfolding and metal-titration data allow for determination of copper (Cu(II) and Cu(I)) and zinc affinities for folded and unfolded azurin polypeptides, respectively.     

Mutagenesis of the redox-active disulfide in mercuric ion reductase: catalysis by mutant enzymes restricted to flavin redox chemistry

Mercuric reductase, a flavoenzyme that possess a redox-active cystine, Cys135Cys140, catalyzes the reduction of Hg(II) to Hg(0) by NADPH. As a probe of mechanism, we have constructed mutants lacking a redox-active disulfide by eliminating Cys135 (Ala135Cys140), Cys140 (Cys135Ala140), or both (Ala135Ala140). Additionally, we have made double mutants that lack Cys135 (Ala135Cys139Cys140) or Cys140 (Cys135Cys139Ala140) but introduce a new Cys in place of Gly139 with the aim of constructing dithiol pairs in the active site that do not form a redox-active disulfide. The resulting mutant enzymes all lack redox-active disulfides and are hence restricted to FAD/FADH2 redox chemistry. Each mutant enzyme possesses unique physical and spectroscopic properties that reflect subtle differences in the FAD microenvironment. These differences are manifested in a 23-nm range in enzyme-bound FAD lambda max values, an 80-nm range in thiolate to flavin charge-transfer absorbance maxima, and a ca. 100-mV range in FAD reduction potential. Preliminary evidence for the Ala135Cys139Cys140 mutant enzyme suggests that this protein forms a disulfide between the two adjacent Cys residues. Hg(II) titration experiments that correlate the extent of charge-transfer quenching with Hg(II) binding indicate that the Ala135Cys140 protein binds Hg(II) with substantially less avidity than does the wild-type enzyme. All mutant mercuric reductases catalyze transhydrogenation and oxygen reduction reactions through obligatory reduced flavin intermediates at rates comparable to or greater than that of the wild-type enzyme. For these activities, there is a linear correlation between log kappa cat and enzyme-bound FAD reduction potential. In a sensitive Hg(II)-mediated enzyme-bound FADH2 reoxidation assay, all mutant enzymes were able to undergo at least one catalytic event at rates 50-1000-fold slower than that of the wild-type enzyme. We have also observed the reduction of Hg(II) by free FADH2. In multiple-turnover assays which monitored the production of Hg(0), two of the mutant enzymes were observed to proceed through at least 30 turnovers at rates ca. 1000-fold slower than that of wild-type mercuric reductase. We conclude that the Cys135 and Cys140 thiols serve as Hg(II) ligands that orient the Hg(II) for subsequent reduction by a reduced flavin intermediate.     

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.     

Identification of a disulfide switch in BsSco, a member of the Sco family of cytochrome c oxidase assembly proteins

BsSco is a membrane-associated protein from Bacillus subtilis characterized by the sequence CXXXCP, which is conserved in yeast and human mitochondrial Sco proteins, and their bacterial homologues. BsSco is involved in the assembly of the Cu(A) center in cytochrome c oxidase and may play a role in the transfer of copper to this site. We have characterized the soluble domain of BsSco by biochemical, spectroscopic, and structural approaches. Soluble BsSco is monomeric in solution, and the two conserved cysteines are involved in an intramolecular cystine bridge. The cystine bridge is easily reduced, and circular dichroism spectroscopy shows no large-scale changes in BsSco's secondary structure upon reduction. The crystal structure of soluble BsSco, determined at 1.7 A resolution, reveals typical elements of a thioredoxin fold. The CXXXCP motif, in which Cys45 and Cys49 are conserved, is located in a turn structure on the surface of the protein. In various native and His135Ala mutant structures, both disulfide-bonded and non-disulfide-bonded forms of CXXXCP are observed. However, despite extensive attempts, copper has not been found near or beyond the CXXXCP motif, a presumptive copper-binding site. Another potential copper binding residue, His135, is located in a highly flexible loop parallel to the CXXXCP loop but is more than 10 A from Cys45 and Cys49. If these three residues are to coordinate copper, a conformational change is necessary. The structural identification of a disulfide switch demonstrates that BsSco has the capability to fill a redox role in Cu(A) assembly.     

Nitrosocyanin, a red cupredoxin-like protein from Nitrosomonas europaea

Nitrosocyanin (NC), a soluble, red Cu protein isolated from the ammonia-oxidizing autotrophic bacterium Nitrosomonas europaea, is shown to be a homo-oligomer of 12 kDa Cu-containing monomers. Oligonucleotides based on the amino acid sequence of the N-terminus and of the C-terminal tryptic peptide were used to sequence the gene by PCR. The translated protein sequence was significantly homologous with the mononuclear cupredoxins such as plastocyanin, azurin, or rusticyanin, the type 1 copper-binding region of nitrite reductase, and the binuclear CuA binding region of N(2)O reductase or cytochrome oxidase. The gene for NC contains a leader sequence indicating a periplasmic location. Optical bands for the red Cu center at 280, 390, 500, and 720 nm have extinction coefficients of 13.9, 7.0, 2.2, and 0.9 mM(-1), respectively. The reduction potential of NC (85 mV vs SHE) is much lower than those for known cupredoxins. Sequence alignments with homologous blue copper proteins suggested copper ligation by Cys95, His98, His103, and Glu60. Ligation by these residues (and a water), a trimeric protein structure, and a cupredoxin beta-barrel fold have been established by X-ray crystallography of the protein [Lieberman, R. L., Arciero, D. M., Hooper, A. B., and Rosenzweig, A. C. (2001) Biochemistry 40, 5674-5681]. EPR spectra of the red copper center indicated a Cu(II) species with a g(parallel) of 2.25 and an A(parallel) of 13.8 mT (144 x 10(-4) cm(-1)), typical of Cu in a type 2 copper environment. NC is the first example of a type 2 copper center in a cupredoxin fold. The open coordination site and type 2 copper suggest a possible catalytic rather than electron transfer function.     

Difference in redox behaviors between copper-binding octarepeat and nonoctarepeat sites in prion protein

We studied the redox behavior of copper-binding sites in prion protein (PrP) to clarify copper’s role in the pathological mechanism underlying prion diseases. We investigated the coordination structures, binding affinities, and redox potentials of copper-binding peptide fragments derived from the N-terminal domain of PrP by density functional theory calculations. We used four models for copper-binding moieties in PrP(60–96): two were derived from the PHGGGWGQ octapeptide repeat region of PrP(60–91), and the others were tripeptide Gly-Thr-His fragments derived from the copper-binding nonoctarepeat site around His96. We found that such PrP-derived copper-binding complexes exhibit conformationally dependent redox behavior; for example, the copper-binding complex derived from the octarepeat region tends to possess high reduction potential for the Cu(II)/Cu(I) couple, exceeding 0 V versus the standard hydrogen electrode, whereas the copper-binding nonoctarepeat model around His96 tends to possess high oxidation potential for the Cu(II)/Cu(III) couple and stabilize the higher-valent Cu(III) state. It is possible that such distinct redox activities of a copper-binding PrP are involved in the mechanism underlying prion diseases.     

A mutagenic study of beta-1,4-galactosyltransferases from Neisseria meningitidis

N-terminal His-tagged recombinant beta-1,4-galactosyltransferase from Neisseria meningitidis was expressed and purified to homogeneity by column chromatography using Ni-NTA resin. Mutations were introduced to investigate the roles of, Ser68, His69, Glu88, Asp90, and Tyr156, which are components of a highly conserved region in recombinant beta-1,4 galactosyltransferase. Also, the functions of three other cysteine residues, Cys65, Cys139, and Cys205, were investigated using site-directed mutagenesis to determine the location of the disulfide bond and the role of the sulfhydryl groups. Purified mutant galactosyltransferases, His69Phe, Glu88Gln and Asp90Asn completely shut down wild-type galactosyltransferase activity (1-3 %). Also, Ser68Ala showed much lower activity than wild-type galactosyltransferase (19 %). However, only the substitution of Tyr156Phe resulted in a slight reduction in galactosyltransferase activity (90 %). The enzyme was found to remain active when the cysteine residues at positions 139 and 205 were replaced separately with serine. However, enzyme reactivity was found to be markedly reduced when Cys65 was replaced with serine (27 %). These results indicate that conserved amino acids such as Cys65, Ser68, His69, Glu88, and Asp90 may be involved in the binding of substrates or in the catalysis of the galactosyltransferase reaction.     

Yeast cox17 solution structure and Copper(I) binding

Cox17 is a 69-residue cysteine-rich, copper-binding protein that has been implicated in the delivery of copper to the Cu(A) and Cu(B) centers of cytochrome c oxidase via the copper-binding proteins Sco1 and Cox11, respectively. According to isothermal titration calorimetry experiments, fully reduced Cox17 binds one Cu(I) ion with a K(a) of (6.15 +/- 5.83) x 10(6) M(-1). The solution structures of both apo and Cu(I)-loaded Cox17 reveal two alpha helices preceded by an extensive, unstructured N-terminal region. This region is reminiscent of intrinsically unfolded proteins. The two structures are very similar overall with residues in the copper-binding region becoming more ordered in Cu(I)-loaded Cox17. Based on the NMR data, the Cu(I) ion has been modeled as two-coordinate with ligation by conserved residues Cys(23) and Cys(26). This site is similar to those observed for the Atx1 family of copper chaperones and is consistent with reported mutagenesis studies. A number of conserved, positively charged residues may interact with complementary surfaces on Sco1 and Cox11, facilitating docking and copper transfer. Taken together, these data suggest that Cox17 is not only well suited to a copper chaperone function but is specifically designed to interact with two different target proteins.     

The essential role of the Cu(II) state of Sco in the maturation of the Cu<Subscript>A</Subscript> center of cytochrome oxidase: evidence from H135Met and H135SeM variants of the <Emphasis Type="Italic">Bacillus subtilis</Emphasis> Sco

Sco is a red copper protein that plays an essential yet poorly understood role in the metalation of the Cu_A center of cytochrome oxidase, and is stable in both the Cu(I) and Cu(II) forms. To determine which oxidation state is important for function, we constructed His135 to Met or selenomethionine (SeM) variants that were designed to stabilize the Cu(I) over the Cu(II) state. H135M was unable to complement a scoΔ strain of Bacillus subtilis, indicating that the His to Met substitution abrogated cytochrome oxidase maturation. The Cu(I) binding affinities of H135M and H135SeM were comparable to that of the WT and 100-fold tighter than that of the H135A variant. The coordination chemistry of the H135M and H135SeM variants was studied by UV/vis, EPR, and XAS spectroscopy in both the Cu(I) and the Cu(II) forms. Both oxidation states bound copper via the S atoms of C45, C49 and M135. In particular, EXAFS data collected at both the Cu and the Se edges of the H135SeM derivative provided unambiguous evidence for selenomethionine coordination. Whereas the coordination chemistry and copper binding affinity of the Cu(I) state closely resembled that of the WT protein, the Cu(II) state was unstable, undergoing autoreduction to Cu(I). H135M also reacted faster with H₂O₂ than WT Sco. These data, when coupled with the complete elimination of function in the H135M variant, imply that the Cu(I) state cannot be the sole determinant of function; the Cu(II) state must be involved in function at some stage of the reaction cycle.     

Met144Ala mutation of the copper-containing nitrite reductase from Alcaligenes xylosoxidans reverses the intramolecular electron transfer

Pulse radiolysis has been employed to investigate the intramolecular electron transfer (ET) between the type 1 (T1) and type 2 (T2) copper sites in the Met144Ala Alcaligenes xylosoxidans nitrite reductase (AxCuNiR) mutant. This mutation increases the reduction potential of the T1 copper center. Kinetic results suggest that the change in driving force has a dramatic influence on the reactivity: The T2Cu(II) is initially reduced followed by ET to T1Cu(II). The activation parameters have been determined and are compared with those of the wild-type (WT) AxCuNiR. The reorganization energy of the T2 site in the latter enzyme was calculated to be 1.6+/-0.2 eV which is two-fold larger than that of the T1 copper center in the WT protein.     

Ab initio modelling of the structure and redox behaviour of copper(I) bound to a His–His model peptide: relevance to the β-amyloid peptide of Alzheimer’s disease

A contributing factor to the pathology of Alzheimer's disease is the generation of reactive oxygen species, most probably a consequence of the beta-amyloid (Abeta) peptide coordinating copper ions. Experimental and theoretical results indicate that His13 and His14 are the two most firmly established ligands in the coordination sphere of Cu(II) bound to Abeta. Abeta1-42 is known to reduce Cu(II) to Cu(I). The Abeta-Cu(II) complex has been shown to catalytically generate H(2)O(2) from reducing agents and O(2). Cu(II) in the presence of Abeta has been reported to have a formal reduction potential of +0.72-0.77 V (vs. the standard hydrogen electrode). Quantum chemical calculations using the B3LYP hybrid density functional method with the 6-31G(d) basis set were performed to model the reduction of previously studied Cu(II) complexes representing the His13-His14 portion of Abeta (Raffa et al. in J. Biol. Inorg. Chem. 10:887-902, 2005). The effects of solvation were accommodated using the CPCM method. The most stable complex between Cu(I) and the model compound, 3-(5-imidazolyl)propionylhistamine (1) involves tricoordinated Cu(I) in a distorted-T geometry, with the Npi of both imidazoles as well as the oxygen of the backbone carbonyl bound to copper. This model would be the most likely representation of a Cu(I) binding site for a His-His peptide in aqueous solution. A variety of possible redox processes are discussed.     

Proximity between Glu126 and Arg144 in the lactose permease of Escherichia coli.

Evidence has been presented [Venkatesan, P., and Kaback, H. R. (1998) Proc. Natl. Acad. Sci. U.S.A. 95, 9802-9807] that Glu126 (helix IV) and Arg144 (helix V) which are critical for substrate binding in the lactose permease of Escherichia coli are charge paired and therefore in close proximity. To test this conclusion more directly, three different site-directed spectroscopic techniques were applied to permease mutants in which Glu126 and/or Arg144 were replaced with either His or Cys residues. (1) Glu126-->His/Arg144-->His permease containing a biotin acceptor domain was purified by monomeric avidin affinity chromatography, and Mn(II) binding was assessed by electron paramagnetic resonance spectroscopy. The mutant protein binds Mn(II) with a KD of about 40 microM at pH 7.5, while no binding is observed at pH 5.5. In addition, no binding is detected with Glu126-->His or Arg144-->His permease. (2) Permease with Glu126-->Cys/Arg144-->Cys and a biotin acceptor domain was purified, labeled with a thiol-specific nitroxide spin-label, and shown to exhibit spin-spin interactions in the frozen state after reconstitution into proteoliposomes. (3) Glu126-->Cys/Arg144-->Cys permease with a biotin acceptor domain was purified and labeled with a thiol-specific pyrene derivative, and fluorescence spectra were obtained after reconstitution into lipid bilayers. An excimer band is observed with the reconstituted E126C/R144C mutant, but not with either single-Cys mutant or when the single-Cys mutants are mixed prior to reconstitution. The results provide strong support for the conclusion that Glu126 (helix IV) and Arg144 (helix V) are in close physical proximity.     

The role of hydrogen bonding at the active site of a cupredoxin: the Phe114Pro azurin variant

The Phe114Pro mutation to the cupredoxin azurin (AZ) leads to a number of structural changes at the active site attributed to deletion of one of the hydrogen bonds to the Cys112 ligand, removal of the bulky phenyl group from the hydrophobic patch of the protein, and steric interactions made by the introduced Pro. The remaining hydrogen bond between the coordinating thiolate and the backbone amide of Asn47 is strengthened. At the type-1 copper site, the Cu(II)-O(Gly45) axial interaction decreases, while the metal moves out of the plane formed by the equatorial His46, Cys112, and His117 ligands, shortening the bond to the axially coordinating Met121. The resulting distorted tetrahedral geometry is distinct from the trigonal bipyramidal arrangement in the wild-type (WT) protein. The unique position of the main S(Cys) --> Cu(II) ligand-to-metal charge-transfer transition in AZ (628 nm) has shifted in the Phe114Pro variant to a value that is more typical for cupredoxins (599 nm). This probably occurs because of the removal of the Phe114-Cys112 hydrogen bond. The Phe114Pro mutation results in a 90 mV decrease in the reduction potential of AZ, and removal of the second hydrogen bond to the Cys ligand seems to be the major cause of this change. The C-terminal His117 ligand does not protonate in the reduced Phe114Pro AZ variant, which suggests that none of the structural features altered by the mutation are responsible for the absence of this effect in the WT protein. Upon reduction, the copper displaces further from the equatorial ligand plane and the Cu-S(Met121) bond length decreases. These changes are larger than those seen in the WT protein and contribute to the order of magnitude decrease in the intrinsic electron-transfer capabilities of the Phe114Pro variant.     

Mutagenesis of the N- and C-terminal cysteine pairs of Tn501 mercuric ion reductase: consequences for bacterial detoxification of mercurials

Mercuric ion reductase (the merA gene product) is a unique member of the class of FAD and redox-active disulfide-containing oxidoreductases by virtue of its ability to reduce Hg(II) to Hg(0) as the last step in bacterial detoxification of mercurials. In addition to the active site redox-active disulfide, formed between Cys135 and Cys140 in Tn501 MerA, the protein products of the three merA gene sequences published to date have two additional conserved pairs of cysteines, one near the N-terminus (Cys10Cys13 in Tn501 MerA) and another near the C-terminus (Cys558Cys559 in Tn501 MerA). Neither of these pairs is found in other members of this enzyme family. To assess the possible roles of these peripheral cysteines in the Hg(II) detoxification pathway, we have constructed and characterized one single mutant, Cys10Ala13, and two double mutants, Ala10Ala13 and Ala558Ala559. The N-terminal mutants are fully functional in vivo as determined by HgCl2 resistance studies, showing the N-terminal cysteine pair to be dispensable. In contrast, the Ala558Ala559 mutant is defective for HgCl2 resistance in vivo and Hg(SR)2 reduction in vitro, thereby implicating Cys558 and/or Cys559 in Hg(II) reduction by the wild-type enzyme. Other activities, such as NADPH/thio-NADP+ transhydrogenation, NADPH oxidation, and DTNB reduction, are unimpaired in this mutant.     

Modulation of activity and substrate binding modes by mutation of single and double subsites +1/+2 and -5/-6 of barley alpha-amylase 1.

Enzymatic properties of barley alpha-amylase 1 (AMY1) are altered as a result of amino acid substitutions at subsites -5/-6 (Cys95-->Ala/Thr) and +1/+2 (Met298-->Ala/Asn/Ser) as well as in the double mutants, Cys95-->Ala/Met298-->Ala/Asn/Ser. Cys95-->Ala shows 176% activity towards insoluble Blue Starch compared to wild-type AMY1, kcat of 142 and 211% towards amylose DP17 and 2-chloro-4-nitrophenyl beta-d-maltoheptaoside (Cl-PNPG7), respectively, but fivefold to 20-fold higher Km. The Cys95-->Thr-AMY1 AMY2 isozyme mimic exhibits the intermediary behaviour of Cys95-->Ala and wild-type. Met298-->Ala/Asn/Ser have slightly higher to slightly lower activity for starch and amylose, whereas kcat and kcat/Km for Cl-PNPG7 are < or = 30% and < or = 10% of wild-type, respectively. The activity of Cys95-->Ala/Met298-->Ala/Asn/Ser is 100-180% towards starch, and the kcat/Km is 15-30%, and 0.4-1.1% towards amylose and Cl-PNPG7, respectively, emphasizing the strong impact of the Cys95-->Ala mutation on activity. The mutants therefore prefer the longer substrates and the specificity ratios of starch/Cl-PNPG7 and amylose/Cl-PNPG7 are 2.8- to 270-fold and 1.2- to 60-fold larger, respectively, than of wild-type. Bond cleavage analyses show that Cys95 and Met298 mutations weaken malto-oligosaccharide binding near subsites -5 and +2, respectively. In the crystal structure Met298 CE and SD (i.e., the side chain methyl group and sulfur atom) are near C(6) and O(6) of the rings of the inhibitor acarbose at subsites +1 and +2, respectively, and Met298 mutants prefer amylose for glycogen, which is hydrolysed with a slightly lower activity than by wild-type. Met298 AMY1 mutants and wild-type release glucose from the nonreducing end of the main-chain of 6"'-maltotriosyl-maltohexaose thus covering subsites -1 to +5, while productive binding of unbranched substrate involves subsites -3 to +3.     

Site-specific hypochlorous acid-induced oxidation of recombinant human myoglobin affects specific amino acid residues and the rate of cytochrome b5-mediated heme reduction

Myeloperoxidase catalyzes the reaction of chloride ions with H₂O₂ to yield hypochlorous acid (HOCl), which can damage proteins. Human myoglobin (HMb) differs from other Mbs by the presence of a cysteine residue at position 110 (Cys110). This study has (i) compared wild-type and a Cys110Ala variant of HMb to assess the influence of Cys110 on HOCl-induced amino acid modification and (ii) determined whether HOCl oxidation of HMb affects the rate of ferric heme reduction by cytochrome b₅. For wild-type HMb (HOCl:Mb ratio of 5:1 mol:mol), Cys110 was preferentially oxidized to a homodimeric or cysteic acid product—sulfenic/sulfinic acids were not detected. At a HOCl:Mb ratio 10:1 mol:mol, methionine (Met) oxidation was detected, and this was enhanced in the Cys110Ala variant. Tryptophan (Trp) oxidation was detected only in the Cys110Ala variant at the highest HOCl dose tested, with oxidation susceptibility following the order Cys > Met > Trp. Tyrosine chlorination was evident only in reactions between HOCl and the Cys110Ala variant and at a longer incubation time (24 h), consistent with the formation via chlorine-transfer reactions from preformed chloramines. HOCl-mediated oxidation of wild-type HMb resulted in a dose-dependent decrease in the observed rate constant for ferric heme reduction (approx two-fold at HOCl:Mb of 10:1 mol:mol). These data indicate that Cys110 influences the oxidation of HMb by HOCl and that oxidation of Cys, Met, and Trp residues is associated with a decrease in the one-electron reduction of ferric HMb by other proteins; such heme-Fe³⁺ reduction is critical to the maintenance of function as an oxygen storage protein in tissues.     

Introduction of a pi-pi interaction at the active site of a cupredoxin: characterization of the Met16Phe Pseudoazurin mutant

The Met16Phe mutant of the type 1 copper protein pseudoazurin (PACu), in which a phenyl ring is introduced close to the imidazole moiety of the His81 ligand, has been characterized. NMR studies indicate that the introduced phenyl ring is parallel to the imidazole group of His81. The mutation has a subtle effect on the position of the two S(Cys)-->Cu(II) ligand-to-metal charge transfer bands in the visible spectrum of PACu(II) and a more significant influence on their intensities resulting in a A(459)/A(598) ratio of 0.31 for Met16Phe as compared to a A(453)/A(594) ratio of 0.43 for wild-type PACu(II) at pH 8. The electron paramagnetic resonance spectrum of the Met16Phe variant is more axial than that of the wild-type protein, and the resonance Raman spectrum of the mutant exhibits subtle differences. A C(gamma)H proton of Met86 exhibits a much smaller hyperfine shift in the paramagnetic (1)H NMR spectrum of Met16Phe PACu(II) as compared to its position in the wild-type protein, which indicates a weaker axial Cu-S(Met86) interaction in the mutant. The Met16Phe mutation results in an approximately 60 mV increase in the reduction potential of PACu. The pK(a) value of the ligand His81 decreases from 4.9 in wild-type PACu(I) to 4.5 in Met16Phe PACu(I) indicating that the pi-pi contact with Phe16 stabilizes the Cu-N(His81) interaction. The Met16Phe variant of PACu has a self-exchange rate constant at pH 7.6 (25 degrees C) of 9.8 x 10(3) M(-)(1) s(-)(1) as compared to the considerably smaller value of 3.7 x 10(3) M(-)(1) s(-)(1) for the wild-type protein under identical conditions. The enhanced electron transfer reactivity of Met16Phe PACu is a consequence of a lower reorganization energy due to additional active site rigidity caused by the pi-pi interaction between His81 and the introduced phenyl ring.     

Effects of the Pathological Q212P Mutation on Human Prion Protein Non-Octarepeat Copper-Binding Site

Prion diseases are a class of fatal neurodegenerative disorders characterized by brain spongiosis, synaptic degeneration, microglia and astrocytes activation, neuronal loss and altered redox control. These maladies can be sporadic, iatrogenic and genetic. The etiological agent is the prion, a misfolded form of the cellular prion protein, PrP(C). PrP(C) interacts with metal ions, in particular copper and zinc, through the octarepeat and non-octarepeat binding sites. The physiological implication of this interaction is still unclear, as is the role of metals in the conversion. Since prion diseases present metal dyshomeostasis and increased oxidative stress, we described the copper-binding site located in the human C-terminal domain of PrP-HuPrP(90-231), both in the wild-type protein and in the protein carrying the pathological mutation Q212P. We used the synchrotron-based X-ray absorption fine structure technique to study the Cu(II) and Cu(I) coordination geometries in the mutant, and we compared them with those obtained using the wild-type protein. By analyzing the extended X-ray absorption fine structure and the X-ray absorption near-edge structure, we highlighted changes in copper coordination induced by the point mutation Q212P in both oxidation states. While in the wild-type protein the copper-binding site has the same structure for both Cu(II) and Cu(I), in the mutant the coordination site changes drastically from the oxidized to the reduced form of the copper ion. Copper-binding sites in the mutant resemble those obtained using peptides, confirming the loss of short- and long-range interactions. These changes probably cause alterations in copper homeostasis and, consequently, in redox control.     

Copper reduction by copper binding proteins and its relation to neurodegenerative diseases

Increasing evidence supports an important role for metals in neurobiology. In fact, copper binding proteins that form bioinorganic complexes are able to display oxidant or anti-oxidant properties, which would impact on neuronal function or in the triggering of neurodegenerative process. Two proteins related to neurodegenerative diseases have been described as copper binding proteins: the amyloid precursor protein (APP), a protein related to Alzheimer's disease, and the Prion protein (PrP), related to Creutzfeldt-Jakob disease. We used different synthetic peptides from APP and PrP sequences in order to evaluate the ability to reduce copper. We observed that APP_135–156, amyloid--peptide (A_1–40), and PrP_59–91 all have copper reducing ability, with the APP_135–156 peptide being more potent than the other fragments. Moreover, we identify His, Cys and Trp residues as key amino acids involved in the copper reduction of A, APP and PrP, respectively. We postulated, that in a cellular context, the interaction of these proteins with copper could be necessary to reduce copper on plasma membrane, possibly presenting Cu(I) to the copper transporter, driving the delivery of this metal to antioxidant enzymes. Moreover, protein-metal complexes could be the catalytic centers for the formation of reactive oxygen species involved in the oxidative damage present both in Alzheimer's and Prion disease.     

Evidence for a copper-binding superfamily of the amyloid precursor protein

The amyloid precursor protein (APP) copper-binding domain (CuBD) has been shown to reduce Cu(II) to Cu(I) and to mediate copper-induced oxidation in vitro. However, little is known about copper binding to the homologous domains of APP and APP family paralogs and orthologs (including amyloid precursor-like proteins from Drosophila melanogaster, Xenopus laevis, and Caenorhabditis elegans) and their effects on Cu-induced oxidation and Cu(I) formation. Here, we show that APP homologues with and without conserved histidine residues at positions 147, 149, and 151 all bind Cu(II). Oxidized peptides were the kinetically favored products of the redox reaction of CuBDs promoting the reduction of Cu(II) to Cu(I). These results reveal a molecular phylogeny-based divergence that has taken place between the ancestral Drosophila APPL and C. elegans APL-1 and the recently evolved APP lineage of CuBDs. Whereas higher species CuBDs have a decreased affinity for Cu(II) and high Cu(II) reducing activities, ancestral CuBDs form very tight binding sites for Cu(II) ions and have low Cu(II) reducing activities. Thus, the APP lineage displays a gain in activity toward promoting Cu(II) reduction and Cu(I) release. The findings suggests that the Cu(II)-binding equilibrium at the phylogenetic stage of Drosophila APPL and C. elegans APL-1 is shifted from the exchangeable Cu(II) pool to the tightly bound, nonexchangeable pool and that ancestral CuBDs may exert antioxidation activities in vivo. The more recently evolved homologues of human APP appear to take advantage of unique redox properties for yet unknown biological functions.