Thermodynamics of azurin folding

The role played by the metal ion in thermodynamics of azurin folding was addressed by studying the thermal denaturation of the apo-form by differential scanning calorimetry (DSC), and by comparing the results with data concerning the holo protein. The thermal unfolding experiments showed that at 25°C the presence of metal ion increases the thermodynamic stability of azurin by 24 kJ mol⁻¹. A comparison between the unfolding and the copper binding free energies allow us to assert that the unfolded polypeptide chain binds copper and subsequently folds into native holo azurin, being this the thermodynamically most favourable process in driving azurin folding.     

ROOM-TEMPERATURE PHOSPHORESCENCE FROM AZURIN DERIVATIVES. PHOSPHORESCENCE QUENCHING IN OXIDIZED NATIVE AZURIN

The tryptophan phosphorescence from a series of derivatives of Pseudomonas aeruginosa azurin has been monitored at 30 degrees C in pH 8.5 buffer solution. The phosphorescence lifetimes fall in the range of 230-270 ms for deoxygenated solutions of derivatives containing Cd(II), Cu(I), Co(II), Ni(II), Hg(II) or apoazurin. A weak signal with a lifetime of ca 130 ms is observed from solutions of oxidized native azurin, but this component is ascribed to a modified form of azurin in solution, i.e. protein heterogeneity, on the basis of the unique sensitivity to quenching by dioxygen. Aside from this minor component, the tryptophan phosphorescence in the Cu(II) protein appears to be fully quenched. The quenching is assigned an electron-transfer mechanism involving transient reduction of the metal center. The same mechanism is deemed to be responsible for fluorescence quenching in oxidized native azurin as well. These observations are of interest because aromatic groups like tryptophan may be conduits for physiological electron-transfer processes involving the copper center.     

Spectroscopy and reactivity of a photogenerated tryptophan radical in a structurally defined protein environment

Near-UV irradiation of structurally characterized [Re(I)(CO)3(1,10-phenanthroline)(Q107H)](W48F/Y72F/H83Q/Y108W)AzM(II) [Az = Pseudomonas aeruginosa azurin, M = Cu, Zn]/[Co(NH3)5Cl]Cl2 produces a tryptophan radical (W108*) with unprecedented kinetic stability. After rapid formation (k = 2.8 x 106 s-1), the radical persists for more than 5 h at room temperature in the folded ReAzM(II) structure. The absorption spectrum of ReAz(W108*)M(II) exhibits maxima at 512 and 536 nm. Oxidation of K4[Mo(CN)8] by ReAz(W108*)Zn(II) places the W108*/W108 reduction potential in the protein above 0.8 V vs NHE.     

Femtomolar Zn(II) affinity in a peptide-based ligand designed to model thiolate-rich metalloprotein active sites

Metal-ligand interactions are critical components of metalloprotein assembly, folding, stability, electrochemistry, and catalytic function. Research over the past 3 decades on the interaction of metals with peptide and protein ligands has progressed from the characterization of amino acid-metal and polypeptide-metal complexes to the design of folded protein scaffolds containing multiple metal cofactors. De novo metalloprotein design has emerged as a valuable tool both for the modular synthesis of these complex metalloproteins and for revealing the fundamental tenets of metalloprotein structure-function relationships. Our research has focused on using the coordination chemistry of de novo designed metalloproteins to probe the interactions of metal cofactors with protein ligands relevant to biological phenomena. Herein, we present a detailed thermodynamic analysis of Fe(II), Co(II), Zn(II), and[4Fe-4S]2(+/+) binding to IGA, a 16 amino acid peptide ligand containing four cysteine residues, H2N-KLCEGG-CIGCGAC-GGW-CONH2. These studies were conducted to delineate the inherent metal-ion preferences of this unfolded tetrathiolate peptide ligand as well as to evaluate the role of the solution pH on metal-peptide complex speciation. The [4Fe-4S]2(+/+)-IGA complex is both an excellent peptide-based synthetic analogue for natural ferredoxins and is flexible enough to accommodate mononuclear metal-ion binding. Incorporation of a single ferrous ion provides the FeII-IGA complex, a spectroscopic model of a reduced rubredoxin active site that possesses limited stability in aqueous buffers. As expected based on the Irving-Williams series and hard-soft acid-base theory, the Co(II) and Zn(II) complexes of IGA are significantly more stable than the Fe(II) complex. Direct proton competition experiments, coupled with determinations of the conditional dissociation constants over a range of pH values, fully define the thermodynamic stabilities and speciation of each MII-IGA complex. The data demonstrate that FeII-IGA and CoII-IGA have formation constant values of 5.0 x 10(8) and 4.2 x 10(11) M-1, which are highly attenuated at physiological pH values. The data also evince that the formation constant for ZnII-IGA is 8.0 x 10(15) M-1, a value that exceeds the tightest natural protein Zn(II)-binding affinities. The formation constant demonstrates that the metal-ligand binding energy of a ZnII(S-Cys)4 site can stabilize a metalloprotein by -21.6 kcal/mol. Rigorous thermodynamic analyses such as those demonstrated here are critical to current research efforts in metalloprotein design, metal-induced protein folding, and metal-ion trafficking.     

Role of the electronic properties of azurin active site in the electron‐transfer process

Electron transfer proteins, such as azurin (a blue copper protein), are promising candidates for the implementation of biomolecular nanoelectronic devices. To understand the details of electron transfer in redox active azurin molecules, we performed plane‐wave pseudo‐potential density functional theory (DFT) calculations of the protein active site in the two possible oxidation states Cu(I) and Cu(II). The ab initio results are used to discuss how the electronic spectrum and wavefunctions may mediate the shuttling of electrons through the copper ion. We find that the Cu‐ligand hybridization is very similar in the two charge states of the metal center, but the energy spectrum changes substantially. This result might indicate important effects of electronic correlations in the redox activity and consequent electron transfer through the Cu site. © 2004 Wiley Periodicals, Inc. Int J Quantum Chem, 2005     

Evidence of zinc superoxide formation in the gas phase: comparisons in behaviour between ligated Zn(I/II) and Cu(I/II) with regard to the attachment of O2 or H2O

Singly and doubly charged atomic ions of zinc and copper have been complexed with pyridine and held in an ion trap. Complexes involving Zn(II) and Cu(I) (3d(10)) display a strong tendency to bind with H(2)O, whilst the Zn(I) (3d(10)4s(1)) complexes exhibit a strong preference for the attachment of O(2). DFT calculations show that this latter result can be interpreted as internal oxidation leading to the formation of superoxide complexes, [Zn(II)O(2)(-)](pyridine)(n), in the gas phase. The calculations also show that the oxidation of Zn(I) to form Zn(II)O(2)(-) is promoted by a mixing of the occupied 4s and vacant 4p orbitals on the metal cation, and that this process is facilitated by the presence of the pyridine ligands.     

Free energy perturbation and molecular dynamics calculations of copper binding to azurin

Free energy perturbation/molecular dynamics simulations have been carried out on copper/azurin systems calculating the binding affinities of copper (II) ion to azurin either in the native or in the unfolded state. In order to test the validity of the strategy adopted for the calculations and to establish what force field is suitable for these kinds of calculations, three different force fields, AMBER, CVFF, and CFF, have been alternatively used for the calculations and the results have been compared with experimental data obtained by spectroscopic titrations of copper (II)/azurin solutions and denaturation experiments. Our findings have pointed out that only CFF gives satisfactory results, thus providing a reliable tool for copper binding simulations in copper protein.     

Binding Selectivity of Methanobactin from <Emphasis Type="Italic">Methylosinus trichosporium</Emphasis> OB3b for Copper(I), Silver(I), Zinc(II), Nickel(II), Cobalt(II), Manganese(II), Lead(II), and Iron(II)

Methanobactin (Mb) from Methylosinus trichosporium OB3b is a member of a class of metal binding peptides identified in methanotrophic bacteria. Mb will selectively bind and reduce Cu(II) to Cu(I), and is thought to mediate the acquisition of the copper cofactor for the enzyme methane monooxygenase. These copper chelating properties of Mb make it potentially useful as a chelating agent for treatment of diseases where copper plays a role including Wilson’s disease, cancers, and neurodegenerative diseases. Utilizing traveling wave ion mobility-mass spectrometry (TWIMS), the competition for the Mb copper binding site from Ag(I), Pb(II), Co(II), Fe(II), Mn(II), Ni(II), and Zn(II) has been determined by a series of metal ion titrations, pH titrations, and metal ion displacement titrations. The TWIMS analyses allowed for the explicit identification and quantification of all the individual Mb species present during the titrations and measured their collision cross-sections and collision-induced dissociation patterns. The results showed Ag(I) and Ni(II) could irreversibly bind to Mb and not be effectively displaced by Cu(I), whereas Ag(I) could also partially displace Cu(I) from the Mb complex. At pH ≈ 6.5, the Mb binding selectivity follows the order Ag(I)≈Cu(I)>Ni(II)≈Zn(II)>Co(II)>>Mn(II)≈Pb(II)>Fe(II), and at pH 7.5 to 10.4 the order is Ag(I)>Cu(I)>Ni(II)>Co(II)>Zn(II)>Mn(II)≈Pb(II)>Fe(II). Breakdown curves of the disulfide reduced Cu(I) and Ag(I) complexes showed a correlation existed between their relative stability and their compact folded structure indicated by their CCS. Fluorescence spectroscopy, which allowed the determination of the binding constant, compared well with the TWIMS analyses, with the exception of the Ni(II) complex.

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Distal metal effects in cobalt porphyrins related to CcO

Cobalt(II) porphyrins were studied to determine the influence of distal site metalation and superstructure upon dioxygen reactivity in active site models of cytochrome c oxidase (CcO). Monometallic, Co(II)(P) complexes when ligated by an axial imidazole react with dioxygen to form reversible Co-superoxide adducts, which were characterized by EPR and resonance Raman (RR). Unexpectedly, certain Co porphyrins with Cu(I) metalated imidazole pickets do not form mu-peroxo Co(III)/Cu(II) products even though the calculated intermetallic distance suggests this is possible. Instead, cobalt-porphyrin-superoxide complexes are obtained with the distal copper remaining as Cu(I). Moreover, distal metals (Cu(I) or Zn(II)) greatly enhance the stability of the dioxygen adduct, such that Co superoxides of bimetallic complexes demonstrate minimal reversibility. The "trapping" of dioxygen by a second metal is attributed to structural and electrostatic changes within the distal pocket upon metalation. EPR evidence suggests that the terminal oxygen in these bimetallic Co-superoxide systems is H-bonded to the NH of an imidazole picket amide linker, which may contribute to enthalpic stabilization of the dioxygen adduct. Stabilization of the dioxygen adduct in these bimetallic systems suggests one possible role for the distal copper in the Fe/Cu bimetallic active site of terminal oxidases, which form a heme-superoxide/copper(I) adduct upon oxygenation.     

A new pyridine-based 12-membered macrocycle functionalised with different fluorescent subunits; coordination chemistry towards Cu(II), Zn(II), Cd(II), Hg(II), and Pb(II)

The coordination chemistry of the new pyridine-based, N2S2-donating 12-membered macrocycle 2,8-dithia-5-aza-2,6-pyridinophane (L1) towards Cu(II), Zn(II), Cd(II), Hg(II), and Pb(II) has been investigated both in aqueous solution and in the solid state. The protonation constants for L1 and stability constants with the aforementioned metal ions have been determined potentiometrically and compared with those of ligand L2, which contains a N-aminopropyl side arm. The measured values show that Hg(II) in water has the highest affinity for both ligands followed by Cu(II), Cd(II), Pb(II), and Zn(II). For each metal ion considered, 1:1 complexes with L1 have also been isolated in the solid state, those of Cu(II) and Zn(II) having also been characterised by X-ray crystallography. In both complexes L1 adopts a folded conformation and the coordination environments around the two metal centres are very similar: four positions of a distorted octahedral coordination sphere are occupied by the donor atoms of the macrocyclic ligand, and the two mutually cis-positions unoccupied by L1 accommodate monodentate NO3- ligands. The macrocycle L1 has then been functionalised with different fluorogenic subunits. In particular, the N-dansylamidopropyl (L3), N-(9-anthracenyl)methyl (L4), and N-(8-hydroxy-2-quinolinyl)methyl (L5) pendant arm derivatives of L1 have been synthesised and their optical response to the above mentioned metal ions investigated in MeCN/H2O (4:1 v/v) solutions.     

Formation of 1 D and 3 D coordination polymers in the solid state induced by mechanochemical and annealing treatments: bis(3-cyano-pentane-2,4-dionato) metal complexes

Bis(3-cyano-pentane-2,4-dionato) (CNacac) metal complex, [M(CNacac)(2)], which acts as both a metal-ion-like and a ligand-like building unit, forms supramolecular structures by self-assembly. Co-grinding of the metal acetates of Mn(II), Co(II), Ni(II), Cu(II) and Zn(II) with CNacacH formed a CNacac complex in all cases: mononuclear complex was formed in the cases of Mn(II), Cu(II) and Zn(II), whereas polymeric ones were formed in the cases of Fe(II), Co(II) and Ni(II). Subsequent annealing converted the mononuclear complexes of Mn(II), Cu(II) and Zn(II) to their corresponding polymers as a result of dehydration of the mononuclear complexes. The resultant Mn(II), Fe(II), Co(II), Ni(II) and Zn(II) polymeric complexes had a common 3 D structure with high thermal stability. In the case of Cu(II), a 1 D polymer was obtained. The Mn(II), Cu(II) and Zn(II) polymeric complexes returned to their original mononuclear complexes on exposure to water vapour but they reverted to the polymeric complexes by re-annealing. Co-grinding of metal chlorides with CNacacH and annealing of the mononuclear CNacac complexes prepared from solution reactions were also examined for comparison. [Mn(CNacac)(2)(H(2)O)(2)], [M(CNacac)(2)(H(2)O)] (M=Cu(II) and Zn(II)) and [M(CNacac)(2)](infinity) (M=Mn(II), Fe(II) and Zn(II)) are new compounds, which clearly indicated the power of the combined mechanochemical/annealing method for the synthesis of varied metal coordination complexes.     

Homo-and heterodinuclear complexes of the tris(catecholamide) derivative of a tetraazamacrocycle with Fe3+, Cu2+ and Zn2+ metal ions

The new ditopic catecholamide 3,7,11-tris-{N-[3,4-(dihydroxybenzoyl)-aminopropyl]} derivative of a 14-membered tetraazamacrocycle containing pyridine (H(6)L(1)) has been synthesized. The protonation constants of (L(1))(6-) and the stability constants of its mono-, homo- and hetero-dinuclear complexes with Fe(3+), Cu(2+) and Zn(2+) metal ions were determined at 298.2 K and ionic strength 0.10 mol dm(-3) in KNO(3). The large overall basicity of the ligand was ascribed to the very high protonation constants of the catecholate groups, and its acid-base behaviour was correlated with the presence of tertiary nitrogen atoms and secondary amide functions. The UV-vis spectrum of the red solution of [FeL(1)](3-) complex exhibits the LMCT band of catecholate to iron(III), and its EPR spectrum revealed a typical isotropic signal of a rhombic distorted ferric centre in a high-spin state and E/D approximately 0.31, both characteristic of a tris-catecholate octahedral environment. The ligand forms with copper(II) and zinc(II) ions mono- and dinuclear protonated complexes and their stability constants were determined, except for the [ML(1)](4-) complexes as the last proton is released at very high pH. Electronic spectroscopic studies of the copper complexes revealed the involvement of catecholate groups in the coordination to the metal centre in the mono- and dinuclear copper(II) complexes. This information together with the determined stability constants indicated that the copper(II) ion can be involved in both types of coordination site of the ligand with comparable binding affinity. The EPR spectrum of [Cu(2)L(1)](2-) showed a well resolved seven-line hyperfine pattern of copper(II) dinuclear species typical of a paramagnetic triplet spin state with weak coupling between the two metal centres. Thermodynamically stable heterodinuclear complexes, [CuFeH(h)L(1)](h-1) (h = 0-3) and [CuZnH(h)L(1)](h-2) (h = 0-4), were formed as expected from a ditopic ligand having two dissimilar coordination sites. At physiological pH, the [CuFeL(1)](-) complex is formed at approximately 100%. The formation of the [CuFeH(h)L(1)](h-1) complexes in solution was supported by electronic spectroscopic measurements. The data indicated the specific coordination of each metal centre at the dissimilar sites of the ligand, the iron(III) bound to the oxygen donors of the catecholate arms and the copper(II) coordinated to the amine donors of the macrocyclic ring. The two metal centres are weakly coupled, due to the fairly large distance between them.     

Synthesis and characterization of a new Schiff base derived from 2,3-diaminopyridine and 5-methoxysalicylaldehyde and its Ni(II), Cu(II) and Zn(II) complexes. Electrochemical and electrocatalytical studies

We describe the synthesis and characterization of a new tetradentate Schiff base ligand obtained from 2,3-diaminopyridine and 5-methoxysalicylaldehyde. This ligand (H₂L) reacted with nickel(II), copper(II), and zinc(II) acetates to give complexes. The ligand and its metal complexes were characterized using analytical, spectral data (UV–vis, IR, and mass spectroscopy), and cyclic voltammetry (CV). The crystal structure of the copper complex was elucidated by X-ray diffraction studies. The electrochemical behavior of these compounds, using CV, revealed that metal centers were distinguished by their intrinsic redox systems, e.g. Ni(II)/Ni(I), Cu(II)/Cu(I), and Zn(II)/Zn(I). Moreover, the electrocatalytic reactions of Ni(II) and Cu(II) complexes catalyze the oxidation of methanol and benzylic alcohol.     

Mechanism for the atomic layer deposition of copper using diethylzinc as the reducing agent: a density functional theory study using gas-phase molecules as a model

We present theoretical studies based on first-principles density functional theory calculations for the possible gas-phase mechanism of the atomic layer deposition (ALD) of copper by transmetalation from common precursors such as Cu(acac)(2), Cu(hfac)(2), Cu(PyrIm(R))(2) with R = (i)Pr and Et, Cu(dmap)(2), and CuCl(2) where diethylzinc acts as the reducing agent. An effect on the geometry and reactivity of the precursors due to differences in electronegativity, steric hindrance, and conjugation present in the ligands was observed. Three reaction types, namely, disproportionation, ligand exchange, and reductive elimination, were considered that together comprise the mechanism for the formation of copper in its metallic state starting from the precursors. A parallel pathway for the formation of zinc in its metallic form was also considered. The model Cu(I) molecule Cu(2)L(2) was studied, as Cu(I) intermediates at the surface play an important role in copper deposition. Through our study, we found that accumulation of an LZnEt intermediate results in zinc contamination by the formation of either Zn(2)L(2) or metallic zinc. Ligand exchange between Cu(II) and Zn(II) should proceed through a Cu(I) intermediate, as otherwise, it would lead to a stable copper molecule rather than copper metal. Volatile ZnL(2) favors the ALD reaction, as it carries the reaction forward.     

Ratiometric fluorescent sensor proteins with subnanomolar affinity for Zn(II) based on copper chaperone domains

The ability to image the concentration of transition metals in living cells in real time is important for further understanding of transition metal homeostasis and its involvement in diseases. The goal of this study was to develop a genetically encoded FRET-based sensor for copper(I) based on the copper-induced dimerization of two copper binding domains involved in human copper homeostasis, Atox1 and the fourth domain of ATP7B (WD4). A sensor has been constructed by linking these copper binding domains to donor and acceptor fluorescent protein domains. Energy transfer is observed in the presence of Cu(I), but the Cu(I)-bridged complex is easily disrupted by low molecular weight thiols such as DTT and glutathione. To our surprise, energy transfer is also observed in the presence of very low concentrations of Zn(II) (10(-)(10) M), even in the presence of DTT. Zn(II) is able to form a stable complex by binding to the cysteines present in the conserved MXCXXC motif of the two copper binding domains. Co(II), Cd(II), and Pb(II) also induce an increase in FRET, but other, physiologically relevant metals are not able to mediate an interaction. The Zn(II) binding properties have been tuned by mutation of the copper-binding motif to the zinc-binding consensus sequence MDCXXC found in the zinc transporter ZntA. The present system allows the molecular mechanism of copper and zinc homeostasis to be studied under carefully controlled conditions in solution. It also provides an attractive platform for the further development of genetically encoded FRET-based sensors for Zn(II) and other transition metal ions.     

Heterodinuclear metal arrangement in a flat macrocycle with two chemically-equivalent metal chelating sites

A phenanthroline-based macrocycle 1 has been newly developed which has two chemically equivalent metal chelating sites within the spatially restricted cavity for dinuclear metal arrangement. The macrocycle 1 reacts with Zn(CF(3)CO(2))(2) or ZnCl(2) to form homodinuclear Zn(II)-complexes. A single-crystal X-ray structural analysis of the resulting Zn(2)1(CF(3)CO(2))(4) determined the complex structure in which two Zn(II) ions are bound by two phenanthroline sites and two CF(3)CO(2)(-) ions bind to each Zn(II) ion in a tetrahedral geometry. Similarly, a homodinuclear Cu(I)-macrocycle was formed from 1 and Cu(CH(3)CN)(4)BF(4). Notably, from 1 and an equimolar mixture of Cu(CH(3)CN)(4)BF(4) and Zn(CF(3)CO(2))(2), a heterodinuclear Cu(I)-Zn(II)-macrocycle was exclusively formed in high yield (>90%) because of the relatively low stability of the dinuclear Cu(I)-macrocycle. A heterodinuclear Ag(I)-Zn(II)-macrocycle was similarly formed with fairly high selectivity from a mixture of Ag(I) and Zn(II) ions. Such selective heterodinuclear metal arrangement was not observed with other combinations of M-Zn(II) (M = Li(I), Mg(II), Pd(II), Hg(II), La(III), and Tb(III)).     

Outer-sphere effects on reduction potentials of copper sites in proteins: the curious case of high potential type 2 C112D/M121E Pseudomonas aeruginosa azurin

Redox and spectroscopic (electronic absorption, multifrequency electron paramagnetic resonance (EPR), and X-ray absorption) properties together with X-ray crystal structures are reported for the type 2 Cu(II) C112D/M121E variant of Pseudomonas aeruginosa azurin. The results suggest that Cu(II) is constrained from interaction with the proximal glutamate; this structural frustration implies a "rack" mechanism for the 290 mV (vs NHE) reduction potential measured at neutral pH. At high pH (～9), hydrogen bonding in the outer coordination sphere is perturbed to allow axial glutamate ligation to Cu(II), with a decrease in potential to 119 mV. These results highlight the role played by outer-sphere interactions, and the structural constraints they impose, in determining the redox behavior of transition metal protein cofactors.     

Electrochemical approach to monitoring metal exchange reaction of acetylacetonate complexes with cadmium, copper and zinc ions

Metal exchange reactions of acetylacetonate complexes with Cd(II), Cu(II) and Zn(II) ions were investigated by using cadmium and copper ion selective electrodes. Changes in the electrode potential and pH of the solutions were monitored upon adding the pertinent metal Zn(II) of the acetylacetonate (AA) complexes. In the reverse system in which a stable Cu-AA complex exists in the solution prior to adding a secondary metal ion (Cd(II) or Zn(II)), no Cu(II) was replaced by either ion. In the systems containing Cd(II) and Zn(II) as a complexed form with AA or as free ions, the exchange reactions were not explained by considering the equilibrium stability constants of the Cd-AA and Zn-AA complexes.     

Catalytic reduction of NO to N2O by a designed heme copper center in myoglobin: implications for the role of metal ions

The effects of metal ions on the reduction of nitric oxide (NO) with a designed heme copper center in myoglobin (F43H/L29H sperm whale Mb, CuBMb) were investigated under reducing anaerobic conditions using UV-vis and EPR spectroscopic techniques as well as GC/MS. In the presence of Cu(I), catalytic reduction of NO to N2O by CuBMb was observed with turnover number of 2 mol NO.mol CuBMb-1.min-1, close to 3 mol NO.mol enzyme-1.min-1 reported for the ba3 oxidases from T. thermophilus. Formation of a His-heme-NO species was detected by UV-vis and EPR spectroscopy. In comparison to the EPR spectra of ferrous-CuBMb-NO in the absence of metal ions, the EPR spectra of ferrous-CuBMb-NO in the presence of Cu(I) showed less-resolved hyperfine splitting from the proximal histidine, probably due to weakening of the proximal His-heme bond. In the presence of Zn(II), formation of a five-coordinate ferrous-CuBMb-NO species, resulting from cleavage of the proximal heme Fe-His bond, was shown by UV-vis and EPR spectroscopic studies. The reduction of NO to N2O was not observed in the presence of Zn(II). Control experiments using wild-type myoglobin indicated no reduction of NO in the presence of either Cu(I) or Zn(II). These results suggest that both the identity and the oxidation state of the metal ion in the CuB center are important for NO reduction. A redox-active metal ion is required to deliver electrons, and a higher oxidation state is preferred to weaken the heme iron-proximal histidine toward a five-coordinate key intermediate in NO reduction.     

Mechanistic studies of Cu(II) binding to amyloid-beta peptides and the fluorescence and redox behaviors of the resulting complexes

Due in large part to the lack of crystal structures of the amyloid-beta (Abeta) peptide and its complexes with Cu(II), Fe(II), and Zn(II), characterization of the metal-Abeta complex has been difficult. In this work, we investigated the complexation of Cu(II) by Abeta through tandem use of fluorescence and electron paramagnetic resonance (EPR) spectroscopies. EPR experiments indicate that Cu(II) bound to Abeta can be reduced to Cu(I) using sodium borohydride and that both Abeta-Cu(II) and Abeta-Cu(I) are chemically stable. Upon reduction of Cu(II) to Cu(I), the Abeta fluorescence, commonly reported to be quenched upon Abeta-Cu(II) complex formation, can be regenerated. The absence of the characteristic tyrosinate peak in the absorption spectra of Abeta-Cu(II) complexes provides evidence that the sole tyrosine residue in Abeta is not one of the four equatorial ligands bound to Cu(II), but remains close to the metal center, and its fluorescence is sensitive to the copper oxidation state and perturbations in the coordination sphere. Further analysis of the quenching and Cu(II) binding behaviors at different Cu(II) concentrations and in the presence of the competing ligand glycine offers evidence supporting the operation of two binding regimes which demonstrate different levels of fluorescence recovery upon addition of the reducing agent. We provide results that suggest the fluorescence quenching is likely caused by charge transfer processes. Thus, by using tyrosine to probe the coordination site, fluorescence spectroscopy provides valuable mechanistic insights into the oxidation state of copper ions bound to Abeta, the binding heterogeneity, and the influence of solution conditions on complex formation.