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This paper reports X-ray chromophorebehavior: the substituents of an X-ray-absorbing atom (here phosphorus) were found to contribute consistent absorptions, in intensity and in relative peak position (transition energy). Linear relations for absolute peak position, as a function of the sum of the phosphorus neighbor-atom electronegativities (Σχ), have been found for ten chromophore peaks: P–F, P–O, P–Cl, P–N, P–CI, P–WI, P–CII(Ph), P–CII(pyr) P–CII(Fc), P–WII. The discovery was made by finding (i) surprisingly rich phosphorus K-edge X-ray absorption spectra [P X-ray absorption spectroscopy (XAS)] for several four-coordinate-phosphorus compounds, including four new ones involving the W(CO)5substituent, (ii) a vastly improved time-dependent (linear-response-based) density-functional-theory prediction method for P XAS which provides unprecedented XAS peak-assignment ability, and (iii) a fresh antibonding-orbital-based interpretive framework. The discovery, that selective X-ray excitation of substituents is possible, opens the door for studies of chemical reactivity implications (X-ray photochemistry), and improved studies of X-ray radiation exposure effects and damage.

Human serum albumin (HSA) is the major copper (Cu) carrier in blood. The majority of previous studies that have investigated Cu interactions with HSA have focused primarily on the Cu(II) oxidation state. Yet, cellular Cu uptake by the human copper transport protein (Ctr1), a plasma membrane-embedded protein responsible for Cu uptake into cells, requires Cu(I). Recent in vitro work has determined that reducing agents, such as the ascorbate present in blood, are sufficient to reduce the Cu(II)HSA complex to form Cu(I)HSA and that Cu(I) is bound to HSA with pM affinity. The biological accessibility of Cu(I)HSA suggests that HSA-bound Cu(I) may be an unappreciated form of Cu cargo and a key player in extracellular Cu trafficking. To better understand Cu trafficking by HSA, we sought to investigate the exchange of Cu(I) from HSA to a model peptide of the Cu-binding ectodomain of Ctr1. In this study, we used X-ray absorption near-edge spectroscopy to show that Cu(I) becomes more highly coordinated as increasing amounts of the Ctr1–14model peptide are added to a solution of Cu(I)HSA. Extended X-ray absorption fine structure (EXAFS) spectroscopy was used to further characterize the interaction of Cu(I)HSA with Ctr1–14by determining the ligands coordinating Cu(I) and their bond lengths. The EXAFS data support that some Cu(I) likely undergoes complete transfer from HSA to Ctr1–14. This finding of HSA interacting with and releasing Cu(I) to an ectodomain model peptide of Ctr1 suggests a mechanism by which HSA delivers Cu(I) to cells under physiological conditions.

Amyloid beta (Aβ) peptides are notorious for their involvement in Alzheimer’s disease (AD), by virtue of their propensity to aggregate to form oligomers, fibrils, and eventually plaques in the brain. Nevertheless, they appear to be essential for correct neurophysiology on the synaptic level and may have additional functions including antimicrobial activity, sealing the blood–brain barrier, promotion of recovery from brain injury, and even tumor suppression. Aβ peptides are also avid copper chelators, and coincidentally copper is significantly dysregulated in the AD brain. Copper (Cu) is released in significant amounts during calcium signaling at the synaptic membrane. Aβ peptides may have a role in maintaining synaptic Cu homeostasis, including as a scavenger for redox-active Cu and as a chaperone for clearing Cu from the synaptic cleft. Here, we employed the Aβ1–16and Aβ4–16peptides as well-established non-aggregating models of major Aβ species in healthy and AD brains, and the Ctr1–14peptide as a model for the extracellular domain of the human cellular copper transporter protein (Ctr1). With these model peptides and a number of spectroscopic techniques, we investigated whether the Cu complexes of Aβ peptides could provide Ctr1 with either Cu(II) or Cu(I). We found that Aβ1–16fully and rapidly delivered Cu(II) to Ctr1–14along the affinity gradient. Such delivery was only partial for the Aβ4–16/Ctr1–14pair, in agreement with the higher complex stability for the former peptide. Moreover, the reaction was very slow and took ca. 40 h to reach equilibrium under the given experimental conditions. In either case of Cu(II) exchange, no intermediate (ternary) species were present in detectable amounts. In contrast, both Aβ species released Cu(I) to Ctr1–14rapidly and in a quantitative fashion, but ternary intermediate species were detected in the analysis of XAS data. The results presented here are the first direct evidence of a Cu(I) and Cu(II) transfer between the human Ctr1 and Aβ model peptides. These results are discussed in terms of the fundamental difference between the peptides’ Cu(II) complexes (pleiotropic ensemble of open structures of Aβ1–16vs the rigid closed-ring system of amino-terminal Cu/Ni binding Aβ4–16) and the similarity of their Cu(I) complexes (both anchored at the tandem His13/His14, bis-His motif). These results indicate that Cu(I) may be more feasible than Cu(II) as the cargo for copper clearance from the synaptic cleft by Aβ peptides and its delivery to Ctr1. The arguments in favor of Cu(I) include the fact that cellular Cu export and uptake proteins (ATPase7A/B and Ctr1, respectively) specifically transport Cu(I), the abundance of extracellular ascorbate reducing agent in the brain, and evidence of a potential associative (hand-off) mechanism of Cu(I) transfer that may mirror the mechanisms of intracellular Cu chaperone proteins.

8-Hydroxyquinolines (8HQs) are a family of lipophilic metal ion chelators that have been used in a range of analytical and pharmaceutical applications over the last 100 years. More recently, CQ (clioquinol; 5-chloro-7-iodo-8-hydroxyquinoline) and PBT2 (5,7-dichloro-2-[(dimethylamino)methyl]-8-hydroxyquinoline) have undergone clinical trials for the treatment of Alzheimer’s disease and Huntington’s disease. Because CQ and PBT2 appear to redistribute metals into cells, these compounds have been redefined as copper and zinc ionophores. Despite the attention surrounding the clinical trials and the clear link between 8HQs and metals, the fundamental solution chemistry of how these compounds bind divalent metals such as copper and zinc, as well as their mechanism(s) of action in mammalian systems, remains poorly understood. In this study, we used a combination of X-ray absorption spectroscopy (XAS), high-energy resolution fluorescence detected (HERFD) XAS, electron paramagnetic resonance (EPR), and UV–visible absorption spectroscopies to investigate the aqueous solution chemistry of a range of 8HQ derivatives. To circumvent the known solubility issues with 8HQ compounds and their complexes with Cu(II), and to avoid the use of abiological organic solvents, we have devised a surfactant buffer system to investigate these Cu(II) complexes in aqueous solution. Our study comprises the first comprehensive investigation of the Cu(II) complexes formed with many 8HQs of interest in aqueous solution, and it provides the first structural information on some of these complexes. We find that halogen substitutions in 8HQ derivatives appear to have little effect on the Cu(II) coordination environment; 5,7-dihalogenated 8HQ conformers all have a pseudo square planar Cu(II) bound by two quinolin-8-olate anions, in agreement with previous studies. Conversely, substituents in the 2-position of the 8HQ moiety appear to cause significant distortions from the typical square-planar-like coordination of most Cu(II)-bis-8HQ complexes, such that the 8HQ moieties in the Cu(II)-bis-8HQ complex are rotated approximately 30–40° apart in a “propeller-like” arrangement.

The amyloid-β (Aβ) peptide is a cleavage product of the amyloid precursor protein and has been implicated as a central player in Alzheimer’s disease. The N-terminal end of Aβ is variable, and different proportions of these variable-length Aβ peptides are present in healthy individuals and those with the disease. The N-terminally truncated form of Aβ starting at position 4 (Aβ4–x) has a His residue as the third amino acid (His6 using the formal Aβ numbering). The N-terminal sequence Xaa-Xaa-His is known as an amino terminal copper and nickel binding motif (ATCUN), which avidly binds Cu(II). This motif is not present in the commonly studied Aβ1–xpeptides. In addition to the ATCUN site, Aβ4–xcontains an additional metal binding site located at the tandem His residues (bis-His at His13 and 14) which is also found in other isoforms of Aβ. Using the ATCUN and bis-His motifs, the Aβ4–xpeptide is capable of binding multiple metal ions simultaneously. We confirm that Cu(II) bound to this particular ATCUN site is redox silent, but the second Cu(II) site is redox active and can be readily reduced with ascorbate. We have employed surrogate metal ions to block copper coordination at the ATCUN or the tandem His site in order to isolate spectral features of the copper coordination environment for structural characterization using extended X-ray absorption fine structure (EXAFS) spectroscopy. This approach reveals that each copper coordination environment is independent in the Cu2Aβ4–xstate. The identification of two functionally different copper binding environments within the Aβ4–xsequence may have important implications for this peptide in vivo.

We report a structural characterization using X-ray absorption spectroscopy of the molybdenum site of Escherichia coliYedY, a novel oxidoreductase related to be the sulfite oxidase family of molybdenum enzymes. We find that the enzyme can exist in MoVand MoIVoxidation states but cannot be readily oxidized to the MoVIform. MoVYedY has molybdenum coordination similar to that of sulfite oxidase, with one MoO at 1.71 Å, three Mo−S at 2.39 Å, and one Mo−OH at 2.09 Å, which elongates to 2.20 Å upon reduction to MoIV, indicating Mo−OH2coordination. The MoVenzyme also possesses a long Mo−O coordination at 2.64 Å, which may be due to oxygen coordination by Asn-45 Oδ, with Mo−Oδapproximately trans to the MoO group. A comparison with sulfite oxidase indicates that YedY possesses a much more uniform Mo−S coordination, with a maximum permitted deviation of less than 0.05 Å. Our results indicate that the YedY active site shows considerable similarity to but also important differences from that of reduced forms of sulfite oxidase.

Human serum albumin (HSA) is a major Cu carrier in human blood and in cerebrospinal fluid. A major assumption is that Cu bound to HSA is in the Cu(II) oxidation state; thus, interactions between HSA and Cu(II) have been intensely investigated for over four decades. HSA has been reported previously to support the reduction of Cu(II) to the Cu(I) oxidation state in the presence of the weak reductant, ascorbate; however, the interactions between HSA and Cu(I) have not been explicitly investigated. Here, we characterize both the apparent affinity of HSA for Cu(I) using solution competition experiments and the coordination structure of Cu(I) bound to HSA using X-ray absorption spectroscopy and in silico modeling. We find that HSA binds to Cu(I) at pH 7.4 with an apparent conditional affinity of KCu(I):HSA= 1014.0using digonal coordination in a structure that is similar to the bis-His coordination modes characterized for amyloid beta (Aβ) and the prion protein. This high affinity and familiar Cu(I) coordination structure suggests that Cu(I) interaction with HSA in human extracellular fluids is unappreciated in the current scientific literature.

Environmental context. Arsenate, in which oxidised arsenic is coordinated to four oxygen atoms, is common in the environment. We have found that a moth larva excretes an unusual form of oxidised arsenic which is bound to six oxygen atoms. Since the chemical groups which give rise to this species are abundant in environmental and biological systems, more research is needed into the possible presence of such six-coordinate complexes in natural systems. Abstract. Arsenate, in which pentavalent arsenic (AsV) is approximately tetrahedrally coordinated by oxygen, is common in biological or environmental systems. Octahedral coordination of AsVby oxygen is known chemically but hitherto has not been observed in natural systems. In an effort to understand the effect of elevated levels of arsenic on insects and other insectivorous animals in the food chain, larvae of the moth bertha armyworm (Mamestra configurataWalker) [Lepidoptera : Noctuidae] were examined under laboratory conditions. Synchrotron X-ray absorption spectroscopy was used to show that the exuvia (shed skin) and frass (fecal matter) contain an unusual AsVspecies six-coordinated by oxygen. The species is modelled as a low pH octahedral chelation complex with vicinal dihydroxyls such as glycerol or catechol. Structural characterisation using extended X-ray absorption fine structure (EXAFS) shows interatomic distances which are more similar to those of the glycerol complex and the near-edge also shows more similarity with the aliphatic chelator. The larvae may be using the six-coordinate AsVspecies as a specific excretory molecule. Since vicinal dihydroxyl species are common, more research is needed into the possible presence of such six-coordinate complexes in natural systems.

The prion protein has garnered considerable interest because of its involvement in prion disease as well as its unresolved cellular function. The octarepeat region in the flexible N-domain is capable of binding copper through multiple coordination modes. Under conditions of low pH and low Cu2+ concentration, the four octarepeats (ORs) cooperatively coordinate a single copper ion. Based on the average structure of the PHGG and GWGQ portions of a copper-free OR2 model from molecular dynamics simulations, the starting structures of the OR4 complex could be constructed by assembling the repeating structure of PHGG and GWGQ fragments. The resulting model contains a preformed site suitable for Cu2+ coordination. Molecular dynamics simulations of Cu2+ bound to the assembled OR4 model (Cu:OR4) reveal a close association of specific Trp and Gly residues with the Cu2+ center. This low Cu2+-occupancy form of prion protein is redox-active and can readily initiate cleavage of the OR region, mediated by reactive oxygen species generated by Cu+. The OR region is known to be required for β-cleavage, as are the Trp residues within the OR region. The β-cleaved form of the prion protein accumulates in amyloid fibrils. Hence, the close approach of Trp and Gly residues to the Cu2+ coordination site in the low Cu2+-occupancy form of the OR region may signal an important interaction for the initiation of prion disease.

Molecular dynamics simulations have been conducted on a model fragment (Ac-PHGGGWGQPHGGGW-NH2) of the prion protein octarepeat domain, both in the Cu2+-bound and metal-free forms. The copper-bound models are based on the consensus structure of the core Cu2+-binding site of an individual octarepeat, relevant to the fully Cu2+-occupied prion protein octarepeat region. The model peptides contain Cu2+ bound through a His imidazole ring and two deprotonated amide N-atoms in the peptide backbone supplied by the following two Gly residues. Both the copper-bound and metal-free models have been simulated with the OPLS all-atom force field with the GROMACS molecular dynamics package. These simulations, with two tandem copper-binding sites, represent the minimum model necessary to observe potential structuring between the copper-binding sites in the octarepeat region. The GWGQ residues constitute a flexible linker region that predominantly adopts a turn, serving to bring adjacent His residues into close proximity. The consequent formation of stable structures demonstrates that the copper-bound octarepeat region allows the copper-coordinating sites to come into van der Waals contact, packing into particular orientations to further stabilize the bend in the GWGQ linker region.

Although Alzheimer’s disease (AD) was first described over a century ago, it remains the leading cause of age-related dementia. Innumerable changes have been linked to the pathology of AD; however, there remains much discord regarding which might be the initial cause of the disease. The “amyloid cascade hypothesis” proposes that the amyloid β (Aβ) peptide is central to disease pathology, which is supported by elevated Aβ levels in the brain before the development of symptoms and correlations of amyloid burden with cognitive impairment. The “metals hypothesis” proposes a role for metal ions such as iron, copper, and zinc in the pathology of AD, which is supported by the accumulation of these metals within amyloid plaques in the brain. Metals have been shown to induce aggregation of Aβ, and metal ion chelators have been shown to reverse this reaction in vitro. 8-Hydroxyqinoline-based chelators showed early promise as anti-Alzheimer’s drugs. Both 5-chloro-7-iodo-8-hydroxyquinoline (CQ) and 5,7-dichloro-2-[(dimethylamino)methyl]-8-hydroxyquinoline (PBT2) underwent unsuccessful clinical trials for the treatment of AD. To gain insight into the mechanism of action of 8HQs, we have investigated the potential interaction of CQ, PBT2, and 5,7-dibromo-8-hydroxyquinoline (B2Q) with Cu(II)-bound Aβ(1–42) using X-ray absorption spectroscopy (XAS), high energy resolution fluorescence detected (HERFD) XAS, and electron paramagnetic resonance (EPR). By XAS, we found CQ and B2Q sequestered ∼83% of the Cu(II) from Aβ(1–42), whereas PBT2 sequestered only ∼59% of the Cu(II) from Aβ(1–42), suggesting that CQ and B2Q have a higher relative Cu(II) affinity than PBT2. From our EPR, it became clear that PBT2 sequestered Cu(II) from a heterogeneous mixture of Cu(II)Aβ(1–42) species in solution, leaving a single Cu(II)Aβ(1–42) species. It follows that the Cu(II) site in this Cu(II)Aβ(1–42) species is inaccessible to PBT2 and may be less solvent-exposed than in other Cu(II)Aβ(1–42) species. We found no evidence to suggest that these 8HQs form ternary complexes with Cu(II)Aβ(1–42).