Metal specificity in DNA damage prevention by sulfur antioxidants

Metals such as Cu^I and Fe^II generate hydroxyl radical (OH) by reducing endogenous hydrogen peroxide (H₂O₂). Because antioxidants can ameliorate metal-mediated oxidative damage, we have quantified the ability of glutathione, a primary intracellular antioxidant, and other biological sulfur-containing compounds to inhibit metal-mediated DNA damage caused hydroxyl radical. In the Cu^I/H₂O₂ system, six sulfur compounds, including both reduced and oxidized glutathione, inhibited DNA damage with IC₅₀ values ranging from 3.4 to 12.4 μM. Glutathione and 3-carboxypropyl disulfide also demonstrated significant antioxidant activity with Fe^II and H₂O₂. Additional gel electrophoresis and UV-vis spectroscopy studies confirm that antioxidant activity for sulfur compounds in the Cu^I system is attributed to metal coordination, a previously unexplored mechanism. The antioxidant mechanism for sulfur compounds in the Fe^II system, however, is unlike that of Cu^I. Our results demonstrate that glutathione and other sulfur compounds are potent antioxidants capable of preventing metal-mediated oxidative DNA damage at well below their biological concentrations. This novel metal-binding antioxidant mechanism may play a significant role in the antioxidant behavior of these sulfur compounds and help refine understanding of glutathione function in vivo.     

Highly flexible O,O′,N ligands and their Fe, Ni, Cu and Zn complexes

Three new chiral ligands bearing an O,O′,N donor set (O_methoxyO_hydroxyN_pyridine) were synthesised and coordinated to Fe^III, Fe^II, Ni^II, Cu^II and Zn^II to yield complexes with the general formula [M(OON)Cl_x]_y. While the pyridine N and the hydroxy O atoms coordinate strongly to all applied metal ions, the methoxy donor seems not to be involved in coordination, although some evidence for a weak interaction between O_Me and the Zn^II were found in NMR spectra. In the bidentate O′,N coordination mode the new ligands exhibit several coordination geometries as analysed in the solid compounds by XRD, EXAFS and EPR and in solution by UV-Vis absorption, cyclic voltammetry, EXAFS, EPR or NMR spectroscopy.     

N-NMR characterization of His residues in and around the active site of FeSOD

We have exploited ¹⁵N-NMR to observe histidine (His) side chains in and around the active site of Fe-containing superoxide dismutase (FeSOD). In the oxidized state, we observe all the non-ligand His side chains and in the reduced state we can account for all the signals in the imidazole spectral region in terms of the non-ligand His′, paramagnetically displaced signals from two backbone amides, and the side chain of glutamine 69 (Gln69). We also observe signals from the His′ that ligate Fe^II. These confirm that neither the Q69H nor the Q69E mutation strongly affects the Fe^II electronic structure, despite the 250 mV and > 660 mV increases in E_m they produce, respectively. In the Q69H mutant, we observe two new signals attributable to the His introduced into the active site in place of Gln69. One corresponds to a protonated N and the other is strongly paramagnetically shifted, to 500 ppm. The strong paramagnetic effects support the existence of an H-bond between His69 and the solvent molecule coordinated to Fe^II, as proposed based on crystallography. Based on previous information that His69 is neutral, we infer that the shifted N is not protonated. Therefore, we propose that this N represents a site of H-bond acceptance from coordinated solvent, representing a reversal of the polarity of this H-bond from that in WT (wild-type) FeSOD protein. We also present evidence that substrate analogs bind to Fe^IISOD outside the Fe^II coordination sphere, affecting Gln69 but without direct involvement of His30.     

A Diatom Ferritin Optimized for Iron Oxidation but Not Iron Storage

Ferritin from the marine pennate diatom Pseudo-nitzschia multiseries (PmFTN) plays a key role in sustaining growth in iron-limited ocean environments. The di-iron catalytic ferroxidase center of PmFTN (sites A and B) has a nearby third iron site (site C) in an arrangement typically observed in prokaryotic ferritins. Here we demonstrate that Glu-44, a site C ligand, and Glu-130, a residue that bridges iron bound at sites B and C, limit the rate of post-oxidation reorganization of iron coordination and the rate at which Fe³⁺ exits the ferroxidase center for storage within the mineral core. The latter, in particular, severely limits the overall rate of iron mineralization. Thus, the diatom ferritin is optimized for initial Fe²⁺ oxidation but not for mineralization, pointing to a role for this protein in buffering iron availability and facilitating iron-sparing rather than only long-term iron storage.     

Coordination complexes of 4-methylimidazole with Zn<Superscript>II</Superscript> and Cu<Superscript>II</Superscript> in gas phase and in water: a DFT study

A quantum chemistry study of mononuclear metal coordination with four 4-methylimidazole ligands (4-MeIm) was investigated. The four complexes [Cu(4-MeIm)₄]²⁺, [Cu(4-MeIm)₄, H₂O]²⁺, [Zn(4-MeIm)₄]²⁺ and [Zn(4-MeIm)₄, H₂O]²⁺ were studied with particular attention to the Nπ or Nτ possible coordinations of the 4-MeIm ring with the metals, using different DFT methods. The results suggest that the Nτ coordination of 4-MeIm ring to Zn^II or Cu^II is more favorable whatever the level of calculation. In contrast, the addition of one water molecule in the first coordination sphere of the metal ions provides five-coordinated complexes showing no Nπ or Nτ preferences. There is good agreement between the DFT-calculated structure and those available experimentally. When metal ions are four-fold coordinated, they adopt a tetrahedral geometry. When Cu^II and Zn^II are five-fold coordinated, highly symmetric structures or intermediate structures are calculated. Similar energies are calculated for different structures, suggesting flat potential energy surfaces. The addition of implicit solvent modifies the calculated first coordination sphere, especially for [Cu(4-MeIm)₄, H₂O]²⁺ structures. The QTAIM and ELF topological analyses of the interaction between Cu^II and the neutral ligands, clearly indicate a dative bonding with a strong ionic character.     

Iron-induced oxidation of (all-E)-β-carotene under model gastric conditions: kinetics,products, and mechanism

The stability of (all-E)-β-carotene toward dietary iron was studied in a mildly acidic (pH 4) micellar solution as a simple model of the postprandial gastric conditions. The oxidation was initiated by free iron (Fe^II, Fe^III) or by heme iron (metmyoglobin, MbFe^III). Fe^II and metmyoglobin were much more efficient than Fe^III at initiating β-carotene oxidation. Whatever the initiator, hydrogen peroxide did not accumulate. Moreover, β-carotene markedly inhibited the conversion of Fe^II into Fe^III. β-Carotene oxidation induced by Fe^II or MbFe^III was maximal with 5–10 eq Fe^II or 0.05–0.1 eq MbFe^III and was inhibited at higher iron concentrations, especially with Fe^II. UPLC/DAD/MS and GC/MS analyses revealed a complex distribution of β-carotene-derived products including Z-isomers, epoxides, and cleavage products of various chain lengths. Finally, the mechanism of iron-induced β-carotene oxidation is discussed. Altogether, our results suggest that dietary iron, especially free (loosely bound) Fe^II and heme iron, may efficiently induce β-carotene autoxidation within the upper digestive tract, thereby limiting its supply to tissues (bioavailability) and consequently its biological activity.     

Multicopper oxidases: a workshop on copper coordination chemistry, electron transfer, and metallophysiology

Multicopper oxidases (MCOs) are unique among copper proteins in that they contain at least one each of the three types of biologic copper sites, type 1, type 2, and the binuclear type 3. MCOs are descended from the family of small blue copper proteins (cupredoxins) that likely arose as a complement to the heme-iron-based cytochromes involved in electron transport; this event corresponded to the aerobiosis of the biosphere that resulted in the conversion of Fe(II) to Fe(III) as the predominant redox state of this essential metal and the solubilization of copper from Cu₂S to Cu(H₂O) _n ²⁺. MCOs are encoded in genomes in all three kingdoms and play essential roles in the physiology of essentially all aerobes. With four redox-active copper centers, MCOs share with terminal copper-heme oxidases the ability to catalyze the four-electron reduction of O₂ to two molecules of water. The electron transfers associated with this reaction are both outer and inner sphere in nature and their mechanisms have been fairly well established. A subset of MCO proteins exhibit specificity for Fe²⁺, Cu⁺, and/or Mn²⁺ as reducing substrates and have been designated as metallooxidases. These enzymes, in particular the ferroxidases found in all fungi and metazoans, play critical roles in the metal metabolism of the expressing organism.     

Both Met(109) and Met(112) are utilized for Cu(II) coordination by the amyloidogenic fragment of the human prion protein at physiological pH

The prion protein is a ubiquitous neuronal membrane protein. Misfolding of the prion protein has been implicated in transmissible spongiform encephalopathies (prion diseases). It has been demonstrated that the human prion protein (PrP) is capable of coordinating at least five Cu^II ions under physiological conditions; four copper binding sites can be found in the octarepeat domain between residues 61 and 91, while another copper binding site can be found in the unstructured “amyloidogenic” domain between residues 91 and 126 PrP(91-126). Herein we expand upon a previous study [J. Shearer, P. Soh, Inorg. Chem. 46 (2007) 710-719] where we demonstrated that the physiologically relevant high affinity Cu^II coordination site within PrP(91-126) is found between residues 106 and 114. It was shown that Cu^II is contained within a square planar (N/O)₃S coordination environment with one His imidazole ligand (H(111)) and one Met thioether ligand (either M(109) or M(112)). The identity of the Met thioether ligand was not identified in that study. In this study we perform a detailed investigation of the Cu^II coordination environment within the PrP fragment containing residues 106-114 (PrP(106-114)) involving optical, X-ray absorption, EPR, and fluorescence spectroscopies in conjunction with electronic structure calculations. By using derivatives of PrP(106-114) with systematic Met → Ile “mutations” we show that the Cu^II coordination environment within PrP(106-114) is actually comprised of a mixture of two major species; one Cu^II(N/O)₃S center with the M(109) thioether coordinated to Cu^II and another Cu^II(N/O)₃S center with the M(112) thioether coordinated to Cu^II. Furthermore, deletion of one or more Met residues from the primary sequence of PrP(106-114) both reduces the Cu^II affinity of the peptide by two to seven fold, and renders the resulting Cu^II metallopeptides redox inactive. The biological implications of these findings are discussed.     

Metal-directed assembly of 1-D and 2-D coordination polymers with fluconazole and dicyanamide co-ligand

The coordination chemistry of a flexible poly(triazolyl)alkane derivative, fluconazole (HFlu), with a series of transition metal ions and dicyanamide (dca) anionic co-ligand has been explored to afford six new metal-organic coordination polymers. Complexes [M(HFlu)₂(dca)₂]_n (M = Mn^II for 1, Fe^II for 2, Co^II for 3, Zn^II for 5, and Cd^II for 6) have the isostructural 1-D double-chain array via bridging fluconazole, whereas [Cu₃(Flu)₂(dca)₄(CH₃OH)₂]_n (4) shows an unusual 2-D layered metal-organic framework with dimeric Cu^II subunits. Notably, both types of coordination patterns are extended into distinct 3-D supramolecular networks via hydrogen-bonding interactions. This result indicates that the choice of metal ion has a significant effect on these polymeric structures as well as the binding modes of the ligands, which is discussed in detail. The Zn^II and Cd^II complexes 5 and 6 display similar fluorescent emissions at 260 nm in the solid state, which essentially are intraligand transitions.     

The impact of spermine competition on the efficacy of DNA-binding Fe(II), Co(II), and Cu(II) complexes of dimeric chromomycin A3

Chromomycin (Chro) forms a 2:1 drug/metal complex through the chelation with Fe(II), Co(II), or Cu(II) ion. The effects of spermine on the interaction of Fe(II), Co(II), and Cu(II) complexes of dimeric Chro with DNA were studied. Circular dichroism (CD) measurements revealed that spermine strongly competed for the Fe(II) and Cu(II) cations in dimeric Chro-DNA complexes, and disrupted the structures of these complexes. However, the DNA-Co^II(Chro)₂ complex showed extreme resistance to spermine-mediated competition for the Co(II) cation. According to surface plasmon resonance (SPR) experiments, a 6 mM concentration of spermine completely abolished the DNA-binding activity of Fe^II(Chro)₂ and Cu^II(Chro)₂ and interfered with the associative binding of Co^II(Chro)₂ complexes to DNA duplexes, but only slightly affected dissociation. In DNA integrity assays, lower concentrations of spermine (1 and 2 mM) promoted DNA strand cleavage by Cu^II(Chro)₂, whereas various concentrations of spermine protected plasmid DNA from damage caused by either Co^II(Chro)₂ or Fe^II(Chro)₂. Additionally, DNA condensation was observed in the reactions of DNA, spermine, and Fe^II(Chro)₂. Despite the fact that Cu^II(Chro)₂ and Fe^II(Chro)₂ demonstrated lower DNA-binding activity than Co^II(Chro)₂ in the absence of spermine, while Cu^II(Chro)₂ and Fe^II(Chro)₂ exhibited greater cytoxicity against HepG2 cells than Co^II(Chro)₂, possibly due to competition of spermine for Fe(II) or Cu(II) in the dimeric Chro complex in the nucleus of the cancer cells. Our results should have significant relevance to future developments in metalloantibiotics for cancer therapy.     

Copper(II) binding properties of hepcidin

Hepcidin is a peptide hormone that regulates the homeostasis of iron metabolism. The N-terminal domain of hepcidin is conserved amongst a range of species and is capable of binding Cu^II and Ni^II through the amino terminal copper–nickel binding motif (ATCUN). It has been suggested that the binding of copper to hepcidin may have biological relevance. In this study we have investigated the binding of Cu^II with model peptides containing the ATCUN motif, fluorescently labelled hepcidin and hepcidin using MALDI-TOF mass spectrometry. As with albumin, it was found that tetrapeptide models of hepcidin possessed a higher affinity for Cu^II than that of native hepcidin. The log K ₁ value of hepcidin for Cu^II was determined as 7.7. Cu^II binds to albumin more tightly than hepcidin (log K ₁ = 12) and in view of the serum concentration difference of albumin and hepcidin, the bulk of kinetically labile Cu^II present in blood will be bound to albumin. It is estimated that the concentration of Cu^II-hepcidin will be less than one femtomolar in normal serum and thus the binding of copper to hepcidin is unlikely to play a role in iron homeostasis. As with albumin, small tri and tetra peptides are poor models for the metal binding properties of hepcidin.     

Does the environment around the H-cluster allow coordination of the pendant amine to the catalytic iron center in [FeFe] hydrogenases? Answers from theory

[FeFe] hydrogenases are H₂-evolving enzymes that feature a diiron cluster in their active site (the [2Fe]_H cluster). One of the iron atoms has a vacant coordination site that directly interacts with H₂, thus favoring its splitting in cooperation with the secondary amine group of a neighboring, flexible azadithiolate ligand. The vacant site is also the primary target of the inhibitor O₂. The [2Fe]_H cluster can span various redox states. The active-ready form (H_ox) attains the Fe^IIFe^I state. States more oxidized than H_ox were shown to be inactive and/or resistant to O₂. In this work, we used density functional theory to evaluate whether azadithiolate-to-iron coordination is involved in oxidative inhibition and protection against O₂, a hypothesis supported by recent results on biomimetic compounds. Our study shows that Fe–N(azadithiolate) bond formation is favored for an Fe^IIFe^II active-site model which disregards explicit treatment of the surrounding protein matrix, in line with the case of the corresponding Fe^IIFe^II synthetic system. However, the study of density functional theory models with explicit inclusion of the amino acid environment around the [2Fe]_H cluster indicates that the protein matrix prevents the formation of such a bond. Our results suggest that mechanisms other than the binding of the azadithiolate nitrogen protect the active site from oxygen in the so-called H _ox ^inact state.     

Spectroscopic characterization of a semi-stable, charge-separated state in Cu-substituted reaction centers from Rhodobacter sphaeroides

In reaction centers from Rhodobacter sphaeroides exposed to continuous illumination in the presence of an inhibitor of the Q_A⁻ to Q_B electron transfer, a semi-stable, charge-separated state was formed with halftimes of formation and decay of several minutes. When the non-heme iron was replaced by Cu²⁺, the decay of the semi-stable, charge-separated state became much slower than in centers with bound Fe²⁺ with about the same rate constant for formation. In Cu²⁺-substituted reaction centers, the semi-stable state was associated with an EPR signal, significantly different from that observed after chemical reduction of the acceptor-side quinone or after illumination at low temperature, but similar to that of an isolated Cu²⁺ in the absence of magnetic interaction. The EPR results, obtained with Cu²⁺-substituted reaction centers, suggest that the slow kinetics of formation and decay of the charge-separated, semi-stable state is associated with a structural rearrangement of the acceptor side and the immediate environment of the metal-binding site.     

Oligonucleotides are potent antioxidants acting primarily through metal ion chelation

We report on a rather unknown feature of oligonucleotides, namely, their potent antioxidant activity. Previously, we showed that nucleotides are potent antioxidants in Fe^II/Cu^I/II–H₂O₂ systems. Here, we explored the potential of 2′-deoxyoligonucleotides as inhibitors of the Fe^II/Cu^I/II-induced ·OH formation from H₂O₂. The oligonucleotides [d(A)_5,7,20; d(T)₂₀; (2′-OMe-A)₅] proved to be highly potent antioxidants with IC₅₀ values of 5–17 or 48–85 μM in inhibiting Fe^II/Cu^I- or Cu^II-induced H₂O₂ decomposition, respectively, thus representing a 40–215-fold increase in potency as compared with Trolox, a standard antioxidant. The antioxidant activity is only weakly dependent on the oligonucleotides’ length or base identity. We analyzed by matrix-assisted laser desorption/ionization time of flight mass spectrometry and ¹H-NMR spectroscopy the composition of the d(A)₅ solution exposed to the aforementioned oxidative conditions for 4 min or 24 h. We concluded that the primary (rapid) inhibition mechanism by oligonucleotides is metal ion chelation and the secondary (slow) mechanism is radical scavenging. We characterized the Cu^I–d(A)₅ and Cu^II–d(A)₇ complexes by ¹H-NMR and ³¹P-NMR or frozen-solution ESR spectroscopy, respectively. Cu^I is probably coordinated to d(A)₅ via N1 and N7 of two adenine residues and possibly also via two phosphate/bridging water molecules. The ESR data suggest Cu^II chelation through two nitrogen atoms of the adenine bases and two oxygen atoms (phosphates or water molecules). We conclude that oligonucleotides at micromolar concentrations prevent Fe^II/Cu^I/II-induced oxidative damage, primarily through metal ion chelation. Furthermore, we propose the use of a short, metabolically stable oligonucleotide, (2′-OMe-A)₅, as a highly potent and relatively long lived (t _1/2 ~ 20 h) antioxidant.     

NO binding to Mn-substituted homoprotocatechuate 2,3-dioxygenase: relationship to O2 reactivity

Iron(II)-containing homoprotocatechuate 2,3-dioxygenase (FeHPCD) activates O₂ to catalyze the aromatic ring opening of homoprotocatechuate (HPCA). The enzyme requires Fe^II for catalysis, but Mn^II can be substituted (MnHPCD) with essentially no change in the steady-state kinetic parameters. Near simultaneous O₂ and HPCA activation has been proposed to occur through transfer of an electron or electrons from HPCA to O₂ through the divalent metal. In O₂ reactions with MnHPCD–HPCA and the 4-nitrocatechol (4NC) complex of the His200Asn (H200N) variant of FeHPCD, this transfer has resulted in the detection of a transient M^III–O₂ ^·? species that is not observed during turnover of the wild-type FeHPCD. The factors governing formation of the M^III–O₂ ^·? species are explored here by EPR spectroscopy using MnHPCD and nitric oxide (NO) as an O₂ surrogate. Both the HPCA and the dihydroxymandelic substrate complexes of MnHPCD bind NO, thus representing the first reported stable MnNO complexes of a nonheme enzyme. In contrast, the free enzyme, the MnHPCD–4NC complex, and the MnH200N and MnH200Q variants with or without HPCA bound do not bind NO. The MnHPCD–ligand complexes that bind NO are also active in normal O₂-linked turnover, whereas the others are inactive. Past studies have shown that FeHPCD and the analogous variants and catecholic ligand complexes all bind NO, and are active in normal turnover. This contrasting behavior may stem from the ability of the enzyme to maintain the approximately 0.8-V difference in the solution redox potentials of Fe^II and Mn^II. Owing to the higher potential of Mn, the formation of the NO adduct or the O₂ adduct requires both strong charge donation from the bound catecholic ligand and additional stabilization by interaction with the active-site His200. The same nonoptimal electronic and structural forces that prevent NO and O₂ binding in MnHPCD variants may lead to inefficient electron transfer from the catecholic substrate to the metal center in variants of FeHPCD during O₂-linked turnover. Accordingly, past studies have shown that intermediate Fe^III species are observed for these mutant enzymes.     

Iron uptake and transfer from ceruloplasmin to transferrin

Background

Dietary and recycled iron are in the Fe^2 + oxidation state. However, the metal is transported in serum by transferrin as Fe^3 +. The multi-copper ferroxidase ceruloplasmin is suspected to be the missing link between acquired Fe^2 + and transported Fe^3 +.

Methods

This study uses the techniques of chemical relaxation and spectrophotometric detection.

Results

Under anaerobic conditions, ceruloplasmin captures and oxidizes two Fe^2 +. The first uptake occurs in domain 6 (< 1 ms) at the divalent iron-binding site. It is accompanied by Fe^2 + oxidation by Cu^2 +_D6. Fe^3 + is then transferred from the binding site to the holding site. Cu⁺_D6 is then re-oxidized by a Cu^2 + of the trinuclear cluster in about 200 ms. The second Fe^2 + uptake and oxidation involve domain 4 and are under the kinetic control of a 200 s change in the protein conformation. With transferrin and in the formed ceruloplasmin–transferrin adduct, two Fe^3 + are transferred from their holding sites to two C-lobes of two transferrins. The first transfer (~ 100 s) is followed by conformation changes (500 s) leading to the release of monoferric transferrin. The second transfer occurs in two steps in the 1000–10,000 second range.

Conclusion

Fe^3 + is transferred after Fe^2 + uptake and oxidation by ceruloplasmin to the C-lobe of transferrin in a protein–protein adduct. This adduct is in a permanent state of equilibrium with all the metal-free or bounded ceruloplasmin and transferrin species present in the medium.

General significance

Ceruloplasmin is a go-between dietary or recycled Fe^2 + and transferrin transported Fe^3 +.     

Mutagenesis of nitrite reductase from Pseudomonas aeruginosa: tyrosine-10 in the c heme domain is not involved in catalysis

In Pseudomonas aeruginosa, conversion of nitrite to NO in dissimilatory denitrification is catalyzed by the enzyme nitrite reductase (NiR), a homodimer containing a covalently bound c heme and a d₁ heme per subunit. We report the purification and characterization of the first single mutant of P. aeruginosa cd₁ NiR in which Tyr¹⁰ has been replaced by Phe; this amino acid was chosen as a possibly important residue in the catalytic mechanism of this enzyme based on the proposal (Fülöp, V., Moir, J.W.B., Ferguson, S.J. and Hajdu, J. (1995) Cell 81, 369–377) that the topologically homologous Tyr²⁵ plays a crucial role in controlling the activity of the cd₁ NiR from Thiosphaera pantotropha. Our results show that in P. aeruginosa NiR substitution of Tyr¹⁰ with Phe has no effect on the activity, optical spectroscopy and electron transfer kinetics of the enzyme, indicating that distal coordination of the Fe³⁺ of the d₁ heme is provided by different side-chains in different species.     

Synthesis, spectroscopy, electrochemistry, and photochemistry of cyano-bridged mixed-valence coordination compounds containing two different types of intervalent charge-transfer bands

The tetranuclear and pentanuclear mixed-valence coordination compounds Na[(NC)₅Fe^II-μ(CN)-Pt^IV(NH₃)₄-μ(NC)-Fe^II(CN)₄-μ(CN)-Ru^III(NH₃)₅], or FePtFeRu, and [Ru^III(NH₃)₅-μ(NC)-Fe^II(CN)₄-μ(CN)-Pt^IV(NH₃)₄-μ(NC)-Fe^II(CN)₄-μ(CN)-Ru^III(NH₃)₅](OSO₂CF₃)₂, or RuFePtFeRu, were synthesized and characterized by IR and UV-Vis spectroscopy, electron microprobe analysis (EPMA), inductively coupled plasma (ICP), and cyclic voltammetry (CV). Both molecules exhibit Fe^II → Pt^IV intervalent charge transfer (IVCT) absorptions in the 400-450 nm range and Fe^II → Ru^III transition(s) between 750 and 950 nm. The energies, intensities, and half-widths of these transitions correspond well with those of model compounds. The cyclic voltammogram of FePtFeRu between 0.00 and 0.90 V versus SCE exhibits two quasi-reversible Fe waves at 0.56 and 0.74 V versus SCE, while that for RuFePtFeRu has only one Fe redox event at 0.72 V versus SCE. When the potential of the working electrode is scanned negative of −0.38 V versus SCE, however, both complexes undergo an ECE (electrochemical-chemical-electrochemical) mechanism whereby the electrochemical reduction of Ru(III) is followed by a double electron transfer to reduce Pt(IV) to Pt(II). Upon reduction to Pt(II), the cyanide bridges break and the complexes dissociate into smaller fragments. Irradiation of the Fe^II → Pt^IV IVCT transition in both compounds leads to a photolysis solution that contains dissociated Fe(II)-Ru(III) as one of its products. Irradiation of the Fe^II → Ru^III IVCT transition yields a similar UV-Vis spectrum, suggesting that the same intermediate is common to both photolysis mechanisms. The implications of this research within the larger context of multiple electron transfer are also discussed.     

Structural and thermodynamical properties of CuII amyloid-β16/28 complexes associated with Alzheimer’s disease

The aggregation of the peptide amyloid-β (Aβ) to form amyloid plaques is a key event in Alzheimer’s disease. It has been shown that Cu^II can bind to soluble Aβ and influence its aggregation properties. Three histidines and the N-terminal amine have been proposed to be involved in its coordination. Here, for the first time, we show isothermal titration calorimetry (ITC) measurements of the Cu^II binding to Aβ16 and Aβ28, models of the soluble Aβ. Moreover, different spectroscopic methods were applied. The studies revealed new insights into these Cu^II–Aβ complexes: (1) ITC showed two Cu^II binding sites, with an apparent K _d of 10⁻⁷ and 10⁻⁵ M, respectively; (2) the high-affinity site has a smaller enthalpic contribution but a larger entropic contribution than the low-affinity binding site; (3) azide did not bind to Cu^II in the higher-affinity binding site, suggesting the absence of a weak, labile ligand; (4) azide could bind to the Cu^II in the low-affinity binding site in Aβ28 but not in Aβ16; (5) ¹H-NMR suggests that the carboxylate of aspartic acid in position 1 is involved in the ligation to Cu^II in the high-affinity binding site; (6) the pK _a of 11.3 of tyrosine in position 10 was not influenced by the binding of 2 equivalents of Cu^II.Electronic Supplementary Material Supplementary material is available to authorized users in the online version of this article at .