Chelator PBT2 Forms a Ternary Cu2+ Complex with ß-Amyloid That Has High Stability but Low Specificity.

The metal chelator PBT2 (5,7-dichloro-2-[(dimethylamino)methyl]-8-hydroxyquinoline) acts as a terdentate ligand capable of forming binary and ternary Cu2+ complexes. It was clinically trialed as an Alzheimer's disease (AD) therapy but failed to progress beyond phase II. The ß-amyloid (Aß) peptide associated with AD was recently concluded to form a unique Cu(Aß) complex that is inaccessible to PBT2. Herein, it is shown that the species ascribed to this binary Cu(Aß) complex in fact corresponds to ternary Cu(PBT2)NImAß complexes formed by the anchoring of Cu(PBT2) on imine nitrogen (NIm) donors of His side chains. The primary site of ternary complex formation is His6, with a conditional stepwise formation constant at pH 7.4 (Kc [M-1]) of logKc = 6.4 ± 0.1, and a second site is supplied by His13 or His14 (logKc = 4.4 ± 0.1). The stability of Cu(PBT2)NImH13/14 is comparable with that of the simplest Cu(PBT2)NIm complexes involving the NIm coordination of free imidazole (logKc = 4.22 ± 0.09) and histamine (logKc = 4.00 ± 0.05). The 100-fold larger formation constant for Cu(PBT2)NImH6 indicates that outer-sphere ligand-peptide interactions strongly stabilize its structure. Despite the relatively high stability of Cu(PBT2)NImH6, PBT2 is a promiscuous chelator capable of forming a ternary Cu(PBT2)NIm complex with any ligand containing an NIm donor. These ligands include histamine, L-His, and ubiquitous His side chains of peptides and proteins in the extracellular milieu, whose combined effect should outweigh that of a single Cu(PBT2)NImH6 complex regardless of its stability. We therefore conclude that PBT2 is capable of accessing Cu(Aß) complexes with high stability but low specificity. The results have implications for future AD therapeutic strategies and understanding the role of PBT2 in the bulk transport of transition metal ions. Given the repurposing of PBT2 as a drug for breaking antibiotic resistance, ternary Cu(PBT2)NIm and analogous Zn(PBT2)NIm complexes may be relevant to its antimicrobial properties.

Ternary Cu2+ Complexes of Human Serum Albumin and Glycyl-l-histidyl-l-lysine.

Human serum albumin (HSA) and the growth factor glycyl-l-histidyl-l-lysine (GHK) bind Cu2+ as part of their normal functions. GHK is found at its highest concentration in the albumin-rich fraction of plasma, leading to speculation that HSA and GHK form a ternary Cu2+ complex. Although preliminary evidence was presented 40 years ago, the structure and stability of such a complex have remained elusive. Here, we show that two ternary Cu(GHK)NImHSA complexes are formed between GHK and the imino nitrogen (NIm) of His side chains of HSA. We identified His3 as one site of ternary complex formation (conditional binding constant cKCu(GHK)NImHis3Cu(GHK) = 2900 M-1 at pH 7.4), with the second site (cKCu(GHK)NImHisXCu(GHK) = 1700 M-1) likely being supplied by either His128 or His510. Together with the established role of HSA as a molecular shuttle in the blood, these complexes may aid the transport of the exchangeable Cu2+ pool and the functional form of GHK.

Intermediate Cu(II)-Thiolate Species in the Reduction of Cu(II)GHK by Glutathione: A Handy Chelate for Biological Cu(II) Reduction.

Gly-His-Lys (GHK) is a tripeptide present in the human bloodstream that exhibits a number of biological functions. Its activity is attributed to the copper-complexed form, Cu(II)GHK. Little is known, however, about the molecular aspects of the mechanism of its action. Here, we examined the reaction of Cu(II)GHK with reduced glutathione (GSH), which is the strongest reductant naturally occurring in human plasma. Spectroscopic techniques (UV-vis, CD, EPR, and NMR) and cyclic voltammetry helped unravel the reaction mechanism. The impact of temperature, GSH concentration, oxygen access, and the presence of ternary ligands on the reaction were explored. The transient GSH-Cu(II)GHK complex was found to be an important reaction intermediate. The kinetic and redox properties of this complex, including tuning of the reduction rate by ternary ligands, suggest that it may provide a missing link in copper trafficking as a precursor of Cu(I) ions, for example, for their acquisition by the CTR1 cellular copper transporter.

The Sub-picomolar Cu2+ Dissociation Constant of Human Serum Albumin.

The apparent affinity of human serum albumin (HSA) for divalent copper has long been the subject of great interest, due to its presumed role as the major Cu2+ -binding ligand in blood and cerebrospinal fluid. Using a combination of electronic absorption, circular dichroism and room-temperature electron paramagnetic resonance spectroscopies, together with potentiometric titrations, we competed the tripeptide GGH against HSA to reveal a conditional binding constant of log cKCuCu(HSA) =13.02±0.05 at pH 7.4. This rigorously determined value of the Cu2+ affinity has important implications for understanding the extracellular distribution of copper.

Ternary Cu(II) Complex with GHK Peptide and Cis-Urocanic Acid as a Potential Physiologically Functional Copper Chelate.

The tripeptide NH2-Gly-His-Lys-COOH (GHK), cis-urocanic acid (cis-UCA) and Cu(II) ions are physiological constituents of the human body and they co-occur (e.g., in the skin and the plasma). While GHK is known as Cu(II)-binding molecule, we found that urocanic acid also coordinates Cu(II) ions. Furthermore, both ligands create ternary Cu(II) complex being probably physiologically functional species. Regarding the natural concentrations of the studied molecules in some human tissues, together with the affinities reported here, we conclude that the ternary complex [GHK][Cu(II)][cis-urocanic acid] may be partly responsible for biological effects of GHK and urocanic acid described in the literature.

The Palladium(II) Complex of Aß4-16 as Suitable Model for Structural Studies of Biorelevant Copper(II) Complexes of N-Truncated Beta-Amyloids.

The Aß4-42 peptide is a major beta-amyloid species in the human brain, forming toxic aggregates related to Alzheimer's Disease. It also strongly chelates Cu(II) at the N-terminal Phe-Arg-His ATCUN motif, as demonstrated in Aß4-16 and Aß4-9 model peptides. The resulting complex resists ROS generation and exchange processes and may help protect synapses from copper-related oxidative damage. Structural characterization of Cu(II)Aß4-x complexes by NMR would help elucidate their biological function, but is precluded by Cu(II) paramagneticism. Instead we used an isostructural diamagnetic Pd(II)-Aß4-16 complex as a model. To avoid a kinetic trapping of Pd(II) in an inappropriate transient structure, we designed an appropriate pH-dependent synthetic procedure for ATCUN Pd(II)Aß4-16, controlled by CD, fluorescence and ESI-MS. Its assignments and structure at pH 6.5 were obtained by TOCSY, NOESY, ROESY, 1H-13C HSQC and 1H-15N HSQC NMR experiments, for natural abundance 13C and 15N isotopes, aided by corresponding experiments for Pd(II)-Phe-Arg-His. The square-planar Pd(II)-ATCUN coordination was confirmed, with the rest of the peptide mostly unstructured. The diffusion rates of Aß4-16, Pd(II)-Aß4-16 and their mixture determined using PGSE-NMR experiment suggested that the Pd(II) complex forms a supramolecular assembly with the apopeptide. These results confirm that Pd(II) substitution enables NMR studies of structural aspects of Cu(II)-Aß complexes.

Oligopeptides Generated by Neprilysin Degradation of ß-Amyloid Have the Highest Cu(II) Affinity in the Whole Aß Family.

The catabolism of ß-amyloid (Aß) is carried out by numerous endopeptidases including neprilysin, which hydrolyzes peptide bonds preceding positions 4, 10, and 12 to yield Aß4-9 and a minor Aß12- x species. Alternative processing of the amyloid precursor protein by ß-secretase also generates the Aß11- x species. All these peptides contain a Xxx-Yyy-His sequence, also known as an ATCUN or NTS motif, making them strong chelators of Cu(II) ions. We synthesized the corresponding peptides, Phe-Arg-His-Asp-Ser-Gly-OH (Aß4-9), Glu-Val-His-His-Gln-Lys-am (Aß11-16), Val-His-His-Gln-Lys-am (Aß12-16), and pGlu-Val-His-His-Gln-Lys-am (pAß11-16), and investigated their Cu(II) binding properties using potentiometry, and UV-vis, circular dichroism, and electron paramagnetic resonance spectroscopies. We found that the three peptides with unmodified N-termini formed square-planar Cu(II) complexes at pH 7.4 with analogous geometries but significantly varied Kd values of 6.6 fM (Aß4-9), 9.5 fM (Aß12-16), and 1.8 pM (Aß11-16). Cyclization of the N-terminal Glu11 residue to the pyroglutamate species pAß11-16 dramatically reduced the affinity (5.8 nM). The Cu(II) affinities of Aß4-9 and Aß12-16 are the highest among the Cu(II) complexes of Aß peptides. Using fluorescence spectroscopy, we demonstrated that the Cu(II) exchange between the Phe-Arg-His and Val-His-His motifs is very slow, on the order of days. These results are discussed in terms of the relevance of Aß4-9, a major Cu(II) binding Aß fragment generated by neprilysin, as a possible Cu(II) carrier in the brain.

Prion protein cleavage fragments regulate adult neural stem cell quiescence through redox modulation of mitochondrial fission and SOD2 expression.

Neurogenesis continues in the post-developmental brain throughout life. The ability to stimulate the production of new neurones requires both quiescent and actively proliferating pools of neural stem cells (NSCs). Actively proliferating NSCs ensure that neurogenic demand can be met, whilst the quiescent pool makes certain NSC reserves do not become depleted. The processes preserving the NSC quiescent pool are only just beginning to be defined. Herein, we identify a switch between NSC proliferation and quiescence through changing intracellular redox signalling. We show that N-terminal post-translational cleavage products of the prion protein (PrP) induce a quiescent state, halting NSC cellular growth, migration, and neurite outgrowth. Quiescence is initiated by the PrP cleavage products through reducing intracellular levels of reactive oxygen species. First, inhibition of redox signalling results in increased mitochondrial fission, which rapidly signals quiescence. Thereafter, quiescence is maintained through downstream increases in the expression and activity of superoxide dismutase-2 that reduces mitochondrial superoxide. We further observe that PrP is predominantly cleaved in quiescent NSCs indicating a homeostatic role for this cascade. Our findings provide new insight into the regulation of NSC quiescence, which potentially could influence brain health throughout adult life.

Interplay between Copper, Neprilysin, and N-Truncation of ß-Amyloid.

Sporadic Alzheimer's disease (AD) is associated with an inefficient clearance of the ß-amyloid (Aß) peptide from the central nervous system. The protein levels and activity of the Zn2+-dependent endopeptidase neprilysin (NEP) inversely correlate with brain Aß levels during aging and in AD. The present study considered the ability of Cu2+ ions to inhibit human recombinant NEP and the role for NEP in generating N-truncated Aß fragments with high-affinity Cu2+ binding motifs that can prevent this inhibition. Divalent copper noncompetitively inhibited NEP ( Ki = 1.0 µM),  while proteolysis of Aß yielded the soluble, Aß4-9 fragment that can bind Cu2+ with femtomolar affinity at pH 7.4. This provides Aß4-9 with the potential to act as a Cu2+ carrier and to mediate its own production by preventing NEP inhibition. Enzyme inhibition at high Zn2+ concentrations ( Ki = 20 µM) further suggests a mechanism for modulating NEP activity, Aß4-9 production, and Cu2+ homeostasis.

Structural Insight into Redox Dynamics of Copper Bound N-Truncated Amyloid-ß Peptides from in Situ X-ray Absorption Spectroscopy.

X-ray absorption spectroscopy of CuII amyloid-ß peptide (Aß) under in situ electrochemical control (XAS-EC) has allowed elucidation of the redox properties of CuII bound to truncated peptide forms. The Cu binding environment is significantly different for the Aß1-16 and the N-truncated Aß4-9, Aß4-12, and Aß4-16 (Aß4-9/12/16) peptides, where the N-truncated sequence (F4R5H6) provides the high-affinity amino-terminal copper nickel (ATCUN) binding motif. Low temperature (ca. 10 K) XAS measurements show the adoption of identical CuII ATCUN-type binding sites (CuIIATCUN) by the first three amino acids (FRH) and a longer-range interaction modeled as an oxygen donor ligand, most likely water, to give a tetragonal pyramid geometry in the Aß4-9/12/16 peptides not previously reported. Both XAS-EC and EPR measurements show that CuII:Aß4-16 can be reduced at mildly reducing potentials, similar to that of CuII:Aß1-16. Reduction of peptides lacking the H13H14 residues, CuII:Aß4-9/12, require far more forcing conditions, with metallic copper the only metal-based reduction product. The observations suggest that reduction of CuIIATCUN species at mild potentials is possible, although the rate of reduction is significantly enhanced by involvement of H13H14. XAS-EC analysis reveals that, following reduction, the peptide acts as a terdentate ligand to CuI (H13, H14 together with the linking amide oxygen atom). Modeling of the EXAFS is most consistent with coordination of an additional water oxygen atom to give a quasi-tetrahedral geometry. XAS-EC analysis of oxidized CuII:Aß4-12/16 gives structural parameters consistent with crystallographic data for a five-coordinate CuIII complex and the CuIIATCUN complex. The structural results suggest that CuII and the oxidation product are both accommodated in an ATCUN-like binding site.

Copper Exchange and Redox Activity of a Prototypical 8-Hydroxyquinoline: Implications for Therapeutic Chelation.

The N-truncated ß-amyloid (Aß) isoform Aß4-x is known to bind Cu(2+) via a redox-silent ATCUN motif with a conditional Kd = 30 fM at pH 7.4. This study characterizes the Cu(2+) interactions and redox activity of Aßx-16 (x = 1, 4) and 2-[(dimethylamino)-methyl-8-hydroxyquinoline, a terdentate 8-hydroxyquinoline (8HQ) with a conditional Kd(CuL) = 35 pM at pH 7.4. Metal transfer between Cu(Aß1-16), CuL, CuL2, and ternary CuL(NIm(Aß)) was rapid, while the corresponding equilibrium between L and Aß4-16 occurred slowly via a metastable CuL(NIm(Aß)) intermediate. Both CuL and CuL2 were redox-silent in the presence of ascorbate, but a CuL(NIm) complex can generate reactive oxygen species. Because the NIm(Aß) ligand will be readily exchangeable with NIm ligands of ubiquitous protein His side chains in vivo, this class of 8HQ ligand could transfer Cu(2+) from inert Cu(Aß4-x) to redox-active CuL(NIm). These findings have implications for the use of terdentate 8HQs as therapeutic chelators to treat neurodegenerative disease.

Resistance of Cu(Aß4-16) to Copper Capture by Metallothionein-3 Supports a Function for the Aß4-42 Peptide as a Synaptic Cu(II) Scavenger.

Aß4-42 is a major species of Aß peptide in the brains of both healthy individuals and those affected by Alzheimer's disease. It has recently been demonstrated to bind Cu(II) with an affinity approximately 3000 times higher than the commonly studied Aß1-42 and Aß1-40 peptides, which are implicated in the pathogenesis of Alzheimer's disease. Metallothionein-3, a protein considered to orchestrate copper and zinc metabolism in the brain and provide antioxidant protection, was shown to extract Cu(II) from Aß1-40 when acting in its native Zn7 MT-3 form. This reaction is assumed to underlie the neuroprotective effect of Zn7 MT-3 against Aß toxicity. In this work, we used the truncated model peptides Aß1-16 and Aß4-16 to demonstrate that the high-affinity Cu(II) complex of Aß4-16 is resistant to Zn7 MT-3 reactivity. This indicates that the analogous complex of the full-length peptide Cu(Aß4-42) will not yield copper to MT-3 in the brain, thus supporting the concept of a physiological role for Aß4-42 as a Cu(II) scavenger in the synaptic cleft.

Cavitation during the protein misfolding cyclic amplification (PMCA) method--The trigger for de novo prion generation?

The protein misfolding cyclic amplification (PMCA) technique has become a widely-adopted method for amplifying minute amounts of the infectious conformer of the prion protein (PrP). PMCA involves repeated cycles of 20 kHz sonication and incubation, during which the infectious conformer seeds the conversion of normally folded protein by a templating interaction. Recently, it has proved possible to create an infectious PrP conformer without the need for an infectious seed, by including RNA and the phospholipid POPG as essential cofactors during PMCA. The mechanism underpinning this de novo prion formation remains unknown. In this study, we first establish by spin trapping methods that cavitation bubbles formed during PMCA provide a radical-rich environment. Using a substrate preparation comparable to that employed in studies of de novo prion formation, we demonstrate by immuno-spin trapping that PrP- and RNA-centered radicals are generated during sonication, in addition to PrP-RNA cross-links. We further show that serial PMCA produces protease-resistant PrP that is oxidatively modified. We suggest a unique confluence of structural (membrane-mimetic hydrophobic/hydrophilic bubble interface) and chemical (ROS) effects underlie the phenomenon of de novo prion formation by PMCA, and that these effects have meaningful biological counterparts of possible relevance to spontaneous prion formation in vivo.

The N†Terminus of α-Synuclein Forms Cu(II)-Bridged Oligomers.

The oligomerization of α-synuclein (αSyn) is one of the defining features of Parkinson's disease. Binding of divalent copper to the N terminus of αSyn has been implicated in both its function and dysfunction. Herein, the molecular details of the Cu(II) /αSyn binding interface have been revealed using a library of synthetic 56-residue αSyn peptides containing site-specific isotopic labels. Using electron paramagnetic resonance spectroscopy, αSyn is shown to coordinate Cu(II) with high affinity via two pH-dependent coordination modes between pH 6.5-8.5. Most remarkably, the data demonstrate that the dominant mode is associated with binding to oligomers (antiparallel dimers and/or cyclic trimers) in which Cu(II) ions occupy intermolecular bridging sites. The findings provide a molecular link between Cu(II) -bound αSyn and its associated quaternary oligomeric structure.

A Functional Role for Aß in Metal Homeostasis? N-Truncation and High-Affinity Copper Binding.

Accumulation of the ß-amyloid (Aß) peptide in extracellular senile plaques rich in copper and zinc is a defining pathological feature of Alzheimer's disease (AD). The Aß1-x (x=16/28/40/42) peptides have been the primary focus of Cu(II) binding studies for more than 15 years; however, the N-truncated Aß4-42 peptide is a major Aß isoform detected in both healthy and diseased brains, and it contains a novel N-terminal FRH sequence. Proteins with His at the third position are known to bind Cu(II) avidly, with conditional log K values at pH 7.4 in the range of 11.0-14.6, which is much higher than that determined for Aß1-x peptides. By using Aß4-16 as a model, it was demonstrated that its FRH sequence stoichiometrically binds Cu(II) with a conditional Kd value of 3×10(-14) M at pH 7.4, and that both Aß4-16 and Aß4-42 possess negligible redox activity. Combined with the predominance of Aß4-42 in the brain, our results suggest a physiological role for this isoform in metal homeostasis within the central nervous system.

Neutron reflectometry studies define prion protein N-terminal peptide membrane binding.

The prion protein (PrP), widely recognized to misfold into the causative agent of the transmissible spongiform encephalopathies, has previously been shown to bind to lipid membranes with binding influenced by both membrane composition and pH. Aside from the misfolding events associated with prion pathogenesis, PrP can undergo various posttranslational modifications, including internal cleavage events. Alpha- and beta-cleavage of PrP produces two N-terminal fragments, N1 and N2, respectively, which interact specifically with negatively charged phospholipids at low pH. Our previous work probing N1 and N2 interactions with supported bilayers raised the possibility that the peptides could insert deeply with minimal disruption. In the current study we aimed to refine the binding parameters of these peptides with lipid bilayers. To this end, we used neutron reflectometry to define the structural details of this interaction in combination with quartz crystal microbalance interrogation. Neutron reflectometry confirmed that peptides equivalent to N1 and N2 insert into the interstitial space between the phospholipid headgroups but do not penetrate into the acyl tail region. In accord with our previous studies, interaction was stronger for the N1 fragment than for the N2, with more peptide bound per lipid. Neutron reflectometry analysis also detected lengthening of the lipid acyl tails, with a concurrent decrease in lipid area. This was most evident for the N1 peptide and suggests an induction of increased lipid order in the absence of phase transition. These observations stand in clear contrast to the findings of analogous studies of Ab and ?-synuclein and thereby support the possibility of a functional role for such N-terminal fragment-membrane interactions.

Redox activity and two-step valence tautomerism in a family of dinuclear cobalt complexes with a spiroconjugated bis(dioxolene) ligand.

A family of dinuclear cobalt complexes with bridging bis(dioxolene) ligands derived from 3,3,3',3'-tetramethyl-1,1'-spirobis(indane-5,5',6,6'-tetrol) (spiroH4) and ancillary ligands based on tris(2-pyridylmethyl)amine (tpa) has been synthesized and characterized. The bis(dioxolene) bridging ligand is redox-active and accessible in the (spiro(cat-cat))(4-), (spiro(SQ-cat))(3-), and (spiro(SQ-SQ))(2-) forms, (cat = catecholate, SQ = semiquinonate). Variation of the ancillary ligand (Mentpa; n = 0-3) by successive methylation of the 6-position of the pyridine rings influences the redox state of the complex, governing the distribution of electrons between the cobalt centers and the bridging ligands. Pure samples of salts of the complexes [Co2(spiro)(tpa)2](2+) (1), [Co2(spiro)(Metpa)2](2+) (2), [Co2(spiro)(Me2tpa)2](2+) (3), [Co2(spiro)(Me3tpa)2](2+) (4), [Co2(spiro)(tpa)2](3+) (5), and [Co2(spiro)(tpa)2](4+) (6) have been isolated, and 1, 4, and 6 have been characterized by single crystal X-ray diffraction. Studies in the solid and solution states using multiple techniques reveal temperature invariant redox states for 1, 2, and 4-6 and provide clear evidence for four different charge distributions: 1 and 2 are Co(III)-(spiro(cat-cat))-Co(III), 4 is Co(II)-(spiro(SQ-SQ))-Co(II), 5 is Co(III)-(spiro(SQ-cat))-Co(III), and 6 is Co(III)-(spiro(SQ-SQ))-Co(III). Of the six complexes, only 3 shows evidence of temperature dependence of the charge distribution, displaying a rare thermally induced two-step valence tautomeric transition from the Co(III)-(spiro(cat-cat))-Co(III) form to Co(II)-(spiro(SQ-cat))-Co(III) and then to Co(II)-(spiro(SQ-SQ))-Co(II) in both solid and solution states. This is the first time a two-step valence tautomeric (VT) transition has been observed in solution. Partial photoinduction of the VT transition is also possible in the solid. Magnetic and spectroscopic studies of 5 and 6 reveal that spiroconjugation of the bis(dioxolene) ligand allows electronic interaction across the spiro bridge, suggesting that thermally activated vibronic coupling between the two cobalt-dioxolene moieties plays a key role in the two-step transition evident for 3.

Mixed ligand Cu2+ complexes of a model therapeutic with Alzheimer's amyloid-ß peptide and monoamine neurotransmitters.

8-Hydroxyquinolines (8HQ) have found widespread application in chemistry and biology due to their ability to complex a range of transition metal ions. The family of 2-substituted 8HQs has been proposed for use in the treatment of Alzheimer's disease (AD). Most notably, the therapeutic PBT2 (Prana Biotechnology Ltd.) has been shown to act as an efficient metal chaperone, disaggregate metal-enriched amyloid plaques comprised of the Aß peptide, inhibit Cu/Aß redox chemistry, and reverse the AD phenotype in transgenic animal models. Yet surprisingly little is known about the molecular interactions at play. In this study, we show that the homologous ligand 2-[(dimethylamino)methyl]-8-hydroxyquinoline (HL) forms a CuL complex with a conditional (apparent) dissociation constant of 0.33 nM at pH 6.9 and is capable of forming ternary Cu(2+) complexes with neurotransmitters including histamine (HA), glutamic acid (Glu), and glycine (Gly), with glutathione disulfide (GSSG), and with histidine (His) side chains of proteins and peptides including the Aß peptide. Our findings suggest a molecular basis for the strong metal chaperone activity of PBT2, its ability to attenuate Cu(2+)/Aß interactions, and its potential to promote neuroprotective and neuroregenerative effects.

The heterogeneous nature of Cu2+ interactions with Alzheimer's amyloid-ß peptide.

Alzheimer's disease (AD) is a neurodegenerative disorder characterized by progressive cognitive and memory impairment. Within the brain, senile plaques, which comprise extracellular deposits of the amyloid-ß peptide (Aß), are the most common pathological feature of AD. A high concentration of Cu(2+) is found within these plaques, which are also areas under oxidative stress. Laboratory work has shown that in vitro Aß will react with Cu(2+) to induce peptide aggregation and the production of reactive oxygen species. As such, this interaction offers a possible explanation for two of the defining pathological features observed in the AD brain: the presence of amyloid plaques, which consist largely of insoluble Aß aggregates, and the abundant oxidative stress therein. Researchers have accordingly put forth the "metals hypothesis" of AD, which postulates that compounds designed to inhibit Cu(2+)/Aß interactions and redistribute Cu(2+) may offer therapeutic potential for treating AD. Characterization of the pH-dependent Cu(2+) coordination of Aß is fundamental to understanding the neurological relevance of Cu(2+)/Aß interactions and aiding the design of new therapeutic agents. In an effort to shed light on the problem, many experimental and theoretical techniques, using a variety of model systems, have been undertaken. The preceding decade has seen numerous conflicting spectroscopic reports concerning the nature of the Cu(2+)/Aß coordination. As the number of studies has grown, the nature of the pH-dependent ligand environment surrounding the Cu(2+) cation has remained a point of contention. In large part, the difficulties can be attributed to inappropriate choices of the model system or to methods that are incapable of quantitatively delineating the presence and identity of multiple Cu(2+) coordination modes. Electron paramagnetic resonance (EPR) is the method of choice for studying paramagnetic metal-protein interactions. With the introduction of site-specific (15)N, (17)O, and (13)C isotopic labels and the application of advanced techniques, EPR is capable of eliminating much of the ambiguity. Recent EPR studies have produced the most definitive picture of the pH-dependent Cu(2+) coordination modes of Aß and enabled researchers to address the inconsistencies present in the literature. In this Account, we begin by briefly introducing the evidence for a role of Cu(2+) in AD as well as the potential physiological and therapeutic implications of that role. We then outline the EPR methodology used to resolve the molecular details of the Cu(2+)/Aß interactions. No drugs are currently available for altering the course of AD, and existing therapies only offer short-term symptomatic relief. This focused picture of the role of Cu(2+) in AD-related plaques offers welcome potential for the development of new methods to combat this devastating disease.

Spectroscopic characterization of the molybdenum cofactor of the sulfane dehydrogenase SoxCD from Paracoccus pantotrophus.

The bacterial sulfane dehydrogenase SoxCD is a distantly related member of the sulfite oxidase (SO) enzyme family that is proposed to oxidize protein-bound sulfide (sulfane) of SoxY as part of a multienzyme mechanism of thiosulfate metabolism. This study characterized the molybdenum cofactor of SoxCD1, comprising the catalytic molybdopterin subunit SoxC and the truncated c-type cytochrome subunit SoxD1. Electron paramagnetic resonance spectroscopy of the Mo(V) intermediate generated by dithionite reduction revealed low- and high-pH species with g and A((95,97)Mo) matrices nearly identical to those of SO, indicating a similar pentacoordinate active site in SoxCD1. However, no sulfite-induced reduction to Mo(V) was detected, nor could a strongly coupled (1)H signal or a phosphate-inhibited species be generated. This indicates that the outer coordination sphere controls substrate binding in SoxCD, permitting access only to protein-bound sulfur via the C-terminal tail of SoxY.