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 K = 10 using 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.

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–x peptides. In addition to the ATCUN site, Aβ4–x contains 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–x peptide 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–x state. The identification of two functionally different copper binding environments within the Aβ4–x sequence may have important implications for this peptide in vivo.

Edited by Ruma Banerjee Copper is critically important for methanotrophic bacteria because their primary metabolic enzyme, particulate methane monooxygenase (pMMO), is copper-dependent. In addition to pMMO, many other copper proteins are encoded in the genomes of methanotrophs, including proteins that contain periplasmic copper-A chaperone (PCu A C) domains. Using bioinformatics analyses, we identified three distinct classes of PCu A C domain-containing proteins in methanotrophs, termed PmoF1, PmoF2, and PmoF3. PCu A C domains from other types of bacteria bind a single Cu(I) ion via an HX n MX 21/22 HXM motif, which is also present in PmoF3, but PmoF1 and PmoF2 lack this motif entirely. Instead, the PCu A C domains of PmoF1 and PmoF2 bind only Cu(II), and PmoF1 binds additional Cu(II) ions in a His-rich extension to its PCu A C domain. Crystal structures of the PmoF1 and PmoF2 PCu A C domains reveal that Cu(II) is coordinated by an N-terminal histidine brace HX 10 H motif. This binding site is distinct from those of previously characterized PCu A C domains but resembles copper centers in CopC proteins and lytic polysaccharide monooxygenase (LPMO) enzymes. Bioinformatics analysis of the entire PCu A C family reveals previously unappreciated diversity, including sequences that contain both the HX n MX 21/22 HXM and HX 10 H motifs, and sequences that lack either set of copper-binding ligands. These findings provide the first characterization of an additional class of copper proteins from methanotrophs, further expand the PCu A C family, and afford new insight into the biological significance of histidine bracemediated copper coordination. Copper enzymes, including cytochrome c oxidase, nitrous oxide reductase, nitrite reductase, superoxide dismutase, and particulate methane monooxygenase (pMMO), 4 play important roles in bacterial metabolism (1). In Gram-negative bacteria, these enzymes are located in the inner membrane and/or periplasm, where copper loading likely takes place via periplasmic chaperone proteins or directly from extracytoplasmic pools (2). One family of periplasmic copper-binding proteins implicated in assembly of the cytochrome c oxidase copper centers is the periplasmic copper-A chaperone (PCu A C) proteins. These proteins have been proposed to play a role in loading the cytochrome c oxidase Cu A and/or Cu B sites (3-8). Genes encoding PCu A Cs are often found neighboring genes encoding Sco1 proteins (3, 9), and transfer of Cu(I) from PCu A C to Sco1 has been demonstrated for PCu A Cs from Streptomyces lividans (7) and Rhodobacter capsulatus (10). However, it remains unclear whether PCu A C is absolutely required for metalation of cytochrome c oxidase because multiple gene disruption studies indicate that it is not critical for cytochrome c oxidase activity (5, 7, 11). In addition, there is evidence that some PCu A C homologs have other functions. For example, the PCu A C homolog AccA is essential for copper nitrite reductase activity in pathogenic Neisseria strains (12). Consistent with a broader ...

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 redoxactive Cu and as a chaperone for clearing Cu from the synaptic cleft. Here, we employed the Aβ 1−16 and Aβ 4−16 peptides as well-established non-aggregating models of major Aβ species in healthy and AD brains, and the Ctr 1−14 peptide 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 Aβ 1−16 fully and rapidly delivered Cu(II) to Ctr 1−14 along the affinity gradient. Such delivery was only partial for the Aβ 4−16 /Ctr 1−14 pair, 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 Ctr 1−14 rapidly 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 Aβ 1−16 vs the rigid closed-ring system of amino-terminal Cu/Ni binding Aβ 4−16 ) 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.

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 Ctr1–14 model 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 Ctr1–14 by 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.