Structure and Affinity of Cu(I) Bound to Human Serum Albumin

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Structure and Affinity of Cu(I) Bound to Human Serum Albumin Madison Sendzik,† M. Jake Pushie,‡ Ewelina Stefaniak,†,§ and Kathryn L. Haas*,† †

Department of Chemistry and Physics, Saint Mary’s College, Notre Dame, Indiana 46556, United States Department of Surgery, University of Saskatchewan, Saskatoon, Saskatchewan S7N 5E5, Canada § Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Pawińskiego 5a, 02-106 Warsaw, Poland ‡

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S Supporting Information *

ABSTRACT: 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 = 1014.0 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.

S

delivering it to organs. Under normal physiological conditions, the concentration of HSA in blood is approximately 0.6 mM.8 HSA is a heart-shaped molecule composed of three homologous domains, referred to as domains I, II, and III. Each domain contains two helical subdomains (referred to as A and B) connected by a random coil.9 The structural arrangement of HSA allows for a number of ligand binding sites, which contribute to its broad range of biological functions, including transport of fatty acids (i.e., myristate), drugs (i.e., ibuprofen and warfarin), thyroid hormones (i.e., thyroxine), and metal ions.10−15 Metal binding to HSA is of both physiological and toxicological relevance; HSA transports essential Cu(II), Zn(II), Ca(II), and Co(II) ions, but can also bind to toxic Ni(II) and Cd(II).10 Furthermore, HSA’s ability to bind to soft Au(I) and Pt(IV) has made it a popular target for a variety of anticancer prodrugs.16,17 To date, four metal binding sites have been identified on HSA. Three of these sites are characterized, and illustrated in Figure 1. The most intensely investigated metal binding site is at the amino terminus (N-terminal site, NTS) where the sequence Asp-Ala-His provides a canonical amino terminal Cu, Ni (ATCUN)18 coordination sequence that binds to Cu(II) with picomolar affinity (1 pM, log KCu(II):HSA) = 12.0) and Ni(II) with micromolar affinity (150 μM).19,20 This site provides four

ophisticated biochemical mechanisms have evolved to control the essential, and potentially toxic, chemistry of Cu.1−3 Multiple biologically accessible oxidation states allow Cu to participate in redox reactions and thus serve as a cofactor in electron transfer reactions that are essential to sustain life.2,4 The redox chemistry that makes Cu indispensable to biology is also that which makes it potentially toxic. Aberrant Cu reactions can generate reactive oxygen species through Fenton-like chemistry,5 resulting in cellular damage or death. Therefore, careful control of Cu redox activity through controlled metalloprotein coordination sites is necessary to support life while avoiding unwanted reactions that promote increased oxidative stress. Extracellular Cu speciation is not well-elucidated, partly due to the complexity and heterogeneity of extracellular fluids and due to the possibility of multiple Cu oxidation states. Although characterization of extracellular Cu speciation is challenging for traditional methods, this complexity also presents exciting opportunities for investigation. It is accepted that most Cu in human blood is not freely exchangeable as it is tightly bound by the ferioxidase enzyme, ceruloplasmin. A small fraction of blood Cu is exchangeable and can be taken up by human cells through transfer to the human copper transport protein, Ctr1, or other cellular Cu acquisition proteins. This exchangeable Cu is bound primarily to human serum albumin (HSA), as well as other low molecular weight molecules.6,7 HSA is the most abundant protein in human blood plasma and cerebrospinal fluid. It is an important factor in regulation and homeostasis of Cu, carrying it in the bloodstream and © 2017 American Chemical Society

Received: September 18, 2017 Published: November 22, 2017 15057

DOI: 10.1021/acs.inorgchem.7b02397 Inorg. Chem. 2017, 56, 15057−15065

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Inorganic Chemistry

Figure 1. Characterized metal binding sites within human serum albumin (HSA). HSA ribbon diagram (from PDB: 5IJF)26 shown with Zn bound to the multimetal binding site (MBS). The small molecule crystal structure of Cu bound to DAHK (CCDC-809109)22 is overlaid at the N-terminus to illustrate the N-terminal binding site (NTS). The chemical structures of Cu bound to the NTS and Zn bound to the MBS are shown in the inset panels. Cys-34 is also highlighted here, but this site does not bind significantly to endogenous metal ions.

nitrogen (4N) donors, composed of the free amino terminus, the histidine imidazole, and two backbone nitrogens in a square planar geometry around a central Cu(II) or Ni(II) ion.18 The NTS ATCUN sequence of HSA can also bind to Co(II) with weaker millimolar affinity.21 Currently, there is no published HSA crystal structure showing the NTS in its metal-bound state; however, the structure of Cu(II) bound to DAHK, a model peptide of HSA’s first four N-terminal amino acids, provides a representative structure (Figure 1).22 A second well-characterized metal binding site, located at the interface of HSA protein domains I and II, is referred to as the multimetal binding site (MBS) for its ability to bind to several different metal ions.10,23 The MBS selectively binds to essential Zn(II) and toxic Cd(II) and can serve as a secondary binding site for Cu(II), Ni(II), and Co(II).10,23 In silico modeling and X-ray absorption spectroscopy (XAS) data of Zn(II) bound to the MBS site at physiological pH are consistent with a 5coordinate Zn(II) bound by four conserved amino acid side chains, His-67(N), Asn-99(O), His-247(N), Asp-249(O), and a solvent water(O).24,25 X-ray crystallographic data has also revealed structural details of Zn(II) bound at the MBS (PDB: 5IJF).26 The Zn(II) coordination revealed in this crystal structure is slightly distorted compared to that proposed from XAS data, probably due the high pH 9 of the crystallization conditions (Figure 1). This difference in observed coordination environments for Zn at different pH values illustrates the flexibility of the HSA structure, which adopts slightly different conformations depending on the presence of bound metals, other ligands, or pH. A third metal binding site is provided by the side-chain thiolate of a reduced cysteine at position 34 (Figure 1 shows Cys-34 in its thiol form). Of the 35 total cysteines in HSA, Cys34 is the only one that does not form an intramolecular disulfide bond. Cys-34 is prone to modification, such as Snitrosylation and formation of intermolecular disulfides with thiol-containing amino acids and peptides; however, approximately 75% of circulating HSA bares a free thiol at this position.27,28 This site is most relevant for Pt and Au therapeutic complexes, but does not interact significantly with other metal ions.10

A fourth site on HSA has been identified as a Cd binding site, with similar affinity as Cd binding to the MBS.23,25 This fourth site is proposed to coordinate Cd with one His ligand and four carboxylates; however, its location is unknown.25,29 The NTS is important for the transport of Cu(II) by HSA, and its ATCUN sequence is a familiar coordination mode employed by several other human Cu-binding proteins.18,30−35 All Cu bound to HSA in blood and other fluids is assumed to be in the Cu(II) oxidation state coordinated in the ATCUN motif at the NTS. Cu(II) bound to ATCUN model peptides is stable in the presence of some oxidizing and reducing agents, but there is compelling evidence that warrants investigation of alternative Cu oxidation states and their interactions with HSA. For example, ATCUN peptides are known to generate relatively stable Cu(III)-ATCUN complexes under physiologically relevant conditions.36 On the other hand, there are reports that HSA may facilitate Cu(II) reduction35,37 by the abundant biological reducing agent, ascorbate,38−40 forming a stable Cu(I)HSA complex. The possibility of HSA interacting with Cu in more than one oxidation state may be relevant in the oxidizing and reducing conditions found in different local extracellular environments, and may be a critical piece of the puzzle in understanding Cu trafficking and biochemistry. Interactions between HSA and either Cu(I) or Cu(III) have been generally unappreciated in the literature and have not been explicitly investigated to our knowledge. Here, we set out to systematically characterize the potential for interactions between Cu(I) and HSA.

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EXPERIMENTAL SECTION

Anaerobic Manipulations. Anaerobic reactions, manipulations, and data collection were performed under 3% H2, 97% N2 atmosphere in a Coy anaerobic chamber (O2 < 3 ppm). Preparation of Stock Solutions. Stock solutions of HSA from two different chemical suppliers (Sigma and Alfa Aesar) and from different origins (recombinant from rice or purified from human serum) were prepared by dissolving the lyophilized protein in Nanopure water. Concentrations were determined using the Edelhoch method41 by diluting 5−10 μL of HSA stock in 1 mL of 8 M urea and measuring the UV−vis absorbance of amino acid side chains (tyrosine, tryptophan, and cysteine) at 276, 278, 280, and 282 nm. Total HSA concentration was determined using the estimated extinction 15058

DOI: 10.1021/acs.inorgchem.7b02397 Inorg. Chem. 2017, 56, 15057−15065

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Inorganic Chemistry coefficients corresponding to these wavelengths.41 Stock solution of GGH (glycylglycyl-L-histidine, Chem-Impex) peptide was prepared by weighing the appropriate amount of GGH into deionized water and standardizing the solution using a predicted extinction coefficient (λ = 214 nm, ε = 7013 M−1 cm−1) for GGH.42 Cu(II) standardized solution (CuSO4, 0.1 M) was purchased from Sigma-Aldrich. Cu(I) solutions were prepared by dissolving tetrakisacetonitrile copper(I) hexafluorophosphate ([Cu(CH3CN)4]PF6, Sigma) in argon-degassed acetonitrile (Sigma) using a Coy anaerobic chamber. Cu(I) solutions were standardized by combining aliquots of Cu(I) stock solution with excess bicinchoninate, (BCA, Sigma) a chromophoric ligand, and measuring UV−vis absorbance of the Cu(I)BCA2 complex (λmax = 562 nm, ε = 7900 M−1)43,44 under anaerobic atmosphere. HEPES (4-(2hydroxyethyl)piperazine-1-ethanesulfonic acid) buffer was prepared by combining appropriate amounts of HEPES and the sodium salt of HEPES (Sigma-Aldrich) in Nanopure water and, where necessary, adjusting the pH to 7.4 using sodium hydroxide. Ascorbate stock solution was prepared in two different ways depending on whether the ascorbate was used in aerobic or anaerobic atmosphere. For solutions handled under anaerobic conditions, the ascorbate was weighed and dissolved in degassed HEPES buffer. Addition of ascorbate did not significantly alter the pH of HEPES buffered solution. Because HEPES buffer may contain trace amounts of metal ions which catalyze oxidation of ascorbate, stock solutions of ascorbate handled in an aerobic atmosphere were prepared in Nanopure water and were combined with other reagents at the time of sample preparation. Reaction of Cu(II)HSA and Cu(II)GGH with Ascorbate at pH 7.4. Cu(II) complexes of HSA and GGH were prepared under ambient aerobic atmosphere and buffered at pH 7.4 with 50 mM HEPES. These solutions were incubated for approximately 10 min and monitored by UV−vis to ensure complete complex formation. Ascorbate solution was added to Cu(II) complex solutions, and UV−vis spectra were monitored immediately following mixing with a CaryBio 100 UV−vis spectrophotometer. Addition of ascorbate did not significantly alter the pH of buffered solutions. Determination of the Cu(I)HSA Binding Constant. Absorption spectra were recorded inside a Coy chamber using 1 cm quartz cuvettes and an SI Photonics (Tucson, AZ) model 420 fiber optic CCD array UV−vis spectrophotometer. Reaction solutions were prepared in 1 mL quartz cuvettes by combining appropriate amounts of stock solutions of [Cu(CH3CN)4PF4], BCA, and HEPES buffer at pH 7.4. The precise concentration of Cu(I)(BCA)2 in each reaction was determined by Beer’s law by measuring the absorbance at 562 nm (ε562 = 7900 M−1 cm−1).43,44 A standardized solution of HSA was titrated into the Cu(I)(BCA)2 solution, while changes in the absorption spectra were recorded after mixing for 5 min to sufficiently allow time for equilibration at each titration point. The decrease in Cu(I)(BCA)2 absorbance at 562 nm is caused by formation of Cu(I)HSA by the chemical equilibrium shown in Scheme 1: Global analysis of the absorption spectra was performed using ReactLab Equilibrium software (Jplus Consulting Ltd.). Further details of global analysis and the fitting model are contained in the Supporting Information accompanying this Article. Cu K-Edge X-ray Absorption Spectroscopy. Solutions of Cu(I) with HSA (2.1 mM HSA, 2 mM CuSO4, 50 mM ascorbate, 50 mM HEPES, 50% v/v glycerol) were prepared under a N2 atmosphere, incubated for at least 30 min to allow Cu binding to HSA and reduction of Cu(II) by ascorbate, and injected into Lucite sample holders with Kapton tape windows and rapidly frozen with liquid N2. HEPES buffer was chosen for its temperature-stable pH buffering capacity.45,46 Although HEPES is known to weakly coordinate Cu(II)47 and to participate in Cu(II) reduction in some cases,48 there is no evidence that HEPES coordinates Cu(I). Steric bulk interferes with HEPES coordination to metal ions,47 and with the bulky protein or BCA ligands employed here, ternary HEPES complexes are further unlikely. Data were collected on beamline 7-3 at the Stanford Synchrotron Radiation Light Source. Samples were maintained at 10 K throughout data collection by use of a closed cycle helium cryostat (Oxford instruments, Abingdon, UK) and a Si(220) double crystal monochromator in the ϕ = 90° orientation and detuned

Scheme 1. Equilibrium Exchange of Cu(I) between Colored Cu(I)(BCA)2 Complex and UV−Vis Silent Cu(I)HSA Complex (Reaction 1)a

a This equilibrium can be used to determine the affinity of Cu(I) for HSA (KCu(I):HSA) using the known formation constant of the Cu(I)(BCA)2 complex (KCu(I):2(BCA) = 1017.3).43

to 50% to achieve effective harmonic rejection. Fluorescence data were collected using a 30-element solid-state Ge detector (Canberra Ltd. Meriden, CT) with a 3 absorption-lengths-thick Ni filter placed between the Soller slits and the sample. Total incoming counts were maintained under 100 kHz per channel. Energies were calibrated against the spectrum of a Cu-foil (first inflection point assigned to 8980.3 eV), recorded simultaneously during data collection. Data were collected in 10 eV steps from 200 to 10 eV below the edge (averaged over 1 s), 0.3 eV steps in the edge region (10 eV below the edge to 30 eV above the edge; averaged over 1 s), and an additional 414 data points with k of 1.02−15.75 Å−1 (averaged over 1−9 s per data point from the beginning to the end of the k-range). Spectra from individual detector channels were inspected prior to data averaging, and known monochromator glitches were removed. Data were worked-up and modeled using EXAFSPAK and FEFF8.25 as described previously.49−53 Curve fitting analysis of multiple scattering models was performed on the unfiltered k3-weighted data over the k-range 1−15 Å−1. Further details of the EXAFS curve fitting model are contained in the Supporting Information accompanying this Article.

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RESULTS Cu(II)HSA Is Reduced by Ascorbate To Form a Cu(I)HSA Complex. Here, we investigated the reactivity of the Cu(II)HSA complex with the abundant biological reducing agent, ascorbate,38−40 and compared it to the reactivity of a Cu(II) complex with the model peptide, GGH, under identical conditions. On the basis of previous reports,35,37 we expected that Cu(II) bound to the ATCUN coordination sequence of HSA may be reduced to Cu(I) by ascorbate due to an unidentified Cu(I) binding site on HSA that can stabilize Cu(I) under aerobic conditions. On the other hand, the GGH model peptide represents a simple ATCUN binding sequence and forms a stable complex with Cu(II).54 GGH does not stabilize Cu(I) under ambient aerobic (containing approximately 20% O2) atmosphere because it does not have strong Cu(I) affinity. Burke et al. have previously investigated the reduction of a closely related complex, Cu(II)GGHG, by ascorbate and found that Cu(I) is not formed under aerobic conditions in the presence of this reductant.36 In testing the hypothesis that Cu(II)HSA, but not Cu(II)GGH, can be reduced by ascorbate, we added varying amounts of ascorbate solution to Cu(II)HSA or Cu(II)GGH in HEPES buffer at pH 7.4 and under ambient aerobic atmosphere (Figure 2). Under these aerobic conditions any Cu(II) reduced to Cu(I) would reoxidize unless the peptide or protein is able to stabilize Cu(I) and slow its 15059

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Figure 2. Cu(II)HSA is reduced to Cu(I)HSA by ascorbate. (A) 1.2 mM apo-HSA, 50 mM HEPES buffer at pH 7.4 combined with 1 mM copper(II) sulfate. Apo-HSA has a broad absorption profile attributed to 17 disulfide bonds. Upon addition of 5 mM sodium ascorbate to Cu(II)HSA, the band at 525 nm decreases over time. (B) Cu(II) bound to the ATCUN site of the model peptide GGH does not form a stable Cu(I) complex. Increase in absorbance below 500 nm is consistent with production of dehydroxyascorbate (see Supporting Information). (C) Comparison of reactions of Cu(II)HSA complex (red squares) and Cu(II)GGH complex (blue circles) upon addition of ascorbate, buffered at pH 7.4 with 50 mM HEPES (representative data from one of three replicate experiments are shown).

Figure 3. HSA removes Cu(I) from Cu(I)(BCA)2 complex. Competition between BCA and HSA for Cu(I) under anaerobic conditions. The reaction solution began at 25 μM Cu(BCA)2, 50 mM HEPES buffer, 0.25% acetonitrile. A titrant solution of HSA was added until the absorbance due to Cu(BCA)2 at 562 nm was no longer detected. The complete removal of Cu(I) from Cu(BCA)2 was accomplished by adding approximately 2 mol equiv of HSA compared to Cu ([HSA]/[Cu] = 2). These data were fit to the model in Scheme 1 and Table S1, yielding the formation constant KCu(I):HSA = 1014.0. See Supporting Information for more detail.

reduction and increased Cu(I)HSA formation are related to the amount of ascorbate in solution (Figure 2C). In contrast, Cu(II) bound to the GGH model peptide does not display a time-dependent decrease in the Cu(II)-ATCUN band (Figure 2B,C). These data support our hypothesis that HSA forms a

oxidation. In the case of Cu(II)HSA, addition of at least 1 mol equiv of ascorbate to Cu results in the reduction of the Cu(II)HSA complex (Figure 2A). This reduction of Cu(II)HSA to Cu(I)HSA is observed as a time-dependent decrease in the Cu(II)-ATCUN d−d band at 525 nm. Faster rate of 15060

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Inorganic Chemistry relatively stable Cu(I)HSA complex, while, as expected, the GGH peptide does not. Cu(I) Binds to HSA with Femtomolar Affinity. If HSA stabilizes Cu(I) under aerobic conditions, as indicated from data shown in Figure 2, then a relatively high affinity constant for formation of this Cu(I)HSA complex is anticipated. On the basis of the facilitation of Cu reduction observed in Figure 2, the Cu(I)HSA formation constant should be in a range similar to that of the apparent Cu(II)HSA formation constant also obtained in HEPES buffer of KCu(II):HSA = 1011.4.19 To determine if this is the case, a competition experiment was employed to measure the apparent affinity of Cu(I) for HSA in HEPES buffer at pH 7.4, as has been done previously to determine Cu(I) affinity for human Ctr1 model peptides.35 The determination of Cu(I)HSA affinity by UV−vis solution competition is enabled by the colorimetric ligand BCA and its known formation constant (see Scheme 1 and Supporting Information, Table S1),43 and is shown in Figure 3. Formation of the Cu(I)HSA complex is observed by the loss of the Cu(I)(BCA)2 band at 562 nm (see Scheme 1 and Figure S1 in Supporting Information). The data shown in Figure 3 are a representative plot of three identical experiments using 99% fatty acid-free HSA purchased from Alfa Aesar (Fischer). Fitting these data to the model shown in Scheme 1 and Table S1 yields a formation constant for the Cu(I)HSA complex of log KCu(I):HSA = 14.0 ± 0.1. This affinity is more than 2 orders of magnitude greater than the constant determined previously for formation of the Cu(II)HSA complex under similar conditions.19 Previous work in the literature has brought to light potential confounding differences in HSA purity and integrity which may affect our results.21,55 We therefore tested additional batches of HSA purchased from a different supplier (Sigma-Aldrich) that were either purified from human serum or produced through recombinant protein expression in rice. Data on three replicate experiments with each of these three sources of HSA indicate no significant differences in the Cu(I) interactions with HSA in the batches from the different manufacturers, or from the recombinant protein compared to that purified from human serum (see Figure S2 in Supporting Information). Structure of Cu(I) Bound to HSA. The Cu K near edge spectrum for Cu(I) in the presence of HSA (Figure 4A) demonstrates that the Cu center is indeed reduced, and exhibits a prominent 1s→4p pre-edge transition, indicative of d10 Cu(I) in a digonal coordination environment. Excitation of a 1s electron requires a change in angular quantum number (Δl ± 1), based on dipole selection rules. The 1s→4p transition is therefore expected to be most intense in highly centrosymmetric environments, where there is little or no mixing of the 4p-orbitals, such as in a linear 2-coordinate complex with chemically identical ligands with comparable Cu−L bond lengths. The probability of this transition is expected to diminish in environments with dissimilar ligands or where the coordination is distorted away from a linear L−Cu−L geometry. Solomon et al. have demonstrated that the Cu(I) pre-edge peak is significantly diminished in 3- and 4-coordinate complexes, whereas the prominent 1s→4p transition for Cu(I)HSA is consistent with 2-coordinate forms of Cu(I).56 The Cu K near edge spectra of several other previously characterized Cu(I) complexes with His imidazole donors are compared to that of the Cu(I) complex with HSA in Figure 4B, including two [CuI(His)2] complexes, one associated with the full-length octarepeat region of the prion protein (which

Figure 4. Cu K near edge spectrum for (A) Cu(I) bound to HSA, and (B) comparison between near edge spectra from Cu(I) bound in 2coordinate His-containing complexes of other biologically relevant species: Model peptides of the prion octrarepeat region are abbreviated as ORn where n = number of repeats in the model peptide, while the model peptide of amyloid beta is abbreviated as Aβ.57

contains four potential His donors),50 and the other associated with the amyloid beta (Aβ) peptide popularly implicated with Alzheimer’s disease.57 Also included for comparison is a Cu(I) complex with a single His-containing peptide fragment from the prion protein which, based on previous DFT-based structure calculations, is proposed to be a [CuI(His)(OH2)] complex.51 The first two major oscillations of the Cu(I) EXAFS spectrum, shown in Figure 5A, demonstrate the multiple beat pattern that is characteristic for imidazole coordination, as has been reported previously.50 The EXAFS spectrum did not demonstrate any contributing Zn contamination, which would appear at ∼13.1 Å−1, and therefore allowed the data to be fitted over the k-range of 1.03−14.97 Å−1, giving an effective resolution (ΔR) of 0.11 Å. Initial EXAFS curve fitting using only single scattering paths gave better fits to the data when a 3coordinate complex or a 2-coordinate complex with significantly different bond lengths was invoked. The primary backscattering peak, centered at ∼1.89 Å (Figure 5B), can be fit with 1 or 2 N atoms. For fits involving 3-coordinate complexes, the third ligand stabilizes at 2.36 Å, which is particularly long for Cu(I). While this might signify a weakly associated atom from HSA that is coincidentally located near the metal center, we note that this atom fits to what appears to be a ringing artifact associated with the primary backscattering 15061

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Table 1. EXAFS Curve Fitting Parameters for CuIHSAa path

N

R

σ2

ΔE0

Single Scattering Models Cu−N 2 1.884(3) 0.0041(2) Cu−N 1 1.858(8) 0.0044(3) Cu−S 1 2.01(1) 0.0076 Cu−N 2 1.888(6) 0.0062(3) Cu−S 1 2.01(1) 0.0107 Cu−N 2 1.882(4) 0.0042(2) Cu−O 1 2.367(8) 0.0042 Multiple Scattering Models Cu−Nimidazole 1 1.863(9) 0.0032(4) Cu−O 1 1.89(1) 0.0047 +15 additional scattering paths Cu−Nimidazole 2 1.887(2) 0.0039(1) +12 additional scattering pathsb

F

−11.4(8) −18 (2)

0.4877 0.5000

−12 (1)

0.4973

−12.3(9)

0.4525

−6.1(4)

0.3273

−2.3(4)

0.2958

a Coordination numbers N, interatomic distances R (Å), Debye− Waller factors (mean-square deviations in interatomic distance) σ2 (Å2), and threshold energy shifts ΔE0 (eV). Values in parentheses are estimated standard deviations in the last digit obtained from the diagonal elements of the covariance matrix. The fit-error function F is

2 where χ(k) defined by F = ∑ k6(χ (k)calcd − χ (k)expt )2 /∑ χ (k)expt are the EXAFS oscillations and k is the photoelectron wavenumber

given by k =

2me ℏ2

(E − E0) . All paths terminate at the originating Cu

atom (not indicated). bFull list of the best-fit multiple scattering model is presented in Supporting Information Table S2.

However, the reduction potential of biological fluids depends on additional factors that vary in local extracellular environments, which include the partial pressure of O2, the concentration of thiols and ascorbate, and other factors.40,59−62 Further, when bound in coordination environments which preferentially stabilize Cu(I) over Cu(II), the potential of the Cu(II)/(I) cycle can be increased. For example, Aβ, a peptide which binds to Cu in extracellular environments, provides a coordination environment for Cu(I) which is composed of a bis-His sequence. Cu(I) bound to this two-coordinate site in Aβ is not oxidized by the presence of atmospheric O2.57,63,64 Another example is the Cu(I) binding site of human Ctr1, which is located in the protein’s extracellular domain. This site slows oxidation of Cu(I) by providing a stable pseudotetrahedral coordination environment, also involving a bis-His motif.49,65 HSA is a major Cu carrier in human extracellular fluids and is assumed to bind exclusively to the Cu(II) oxidation state. Whether HSA can also bind to Cu(I) has not been explicitly investigated until now, although there have been at least two recent reports in which Cu(I) binding to HSA is suggested by the observation that Cu(II)HSA complexes can be reduced to form stable Cu(I)HSA complexes under both anaerobic and aerobic conditions.35,37 These previous observations for HSA are similar to phenomena observed for Cu(I) coordination by Aβ and Ctr1 polypeptides mentioned above and may indicate that HSA possesses a Cu(I) binding site that can stabilize Cu(I) in aerobic conditions. Ctr1 and Aβ peptides both stabilize Cu(I) through bis-His coordination. Unlike Ctr1 and Aβ, HSA possesses no bis-His sequence (no two His that are adjacent to each other in sequence); however, it does possess several His side chains that are close in space in the folded protein. These interesting parallels between Ctr1, Aβ, and HSA beg the questions regarding whether HSA interacts with Cu(I) using

Figure 5. Results of the EXAFS curve fitting for Cu(I)HSA. Fits for the EXAFS (A) and EXAFS Fourier transform (B) are overlaid on the experimental data. The best fit is obtained using a [CuI(His)2] multiple scattering model (inset) with both Cu−N bond distances set to 1.889 Å with a bond angle of 180°. The Fourier transform is phasecorrected for Cu−N.

peak in the Fourier transform. As with any such result, chemical rationale must be employed to judge whether such a fit is real or not.51 We note that while it may be feasible to have such a long-range interaction, the presence of an additional atom would greatly perturb the otherwise centrosymmetric environment of the Cu(I) center and lead to a significantly different near edge spectrum, with a greatly diminished 1s → 4p peak intensity.56 Furthermore, attempts to fit a Cu−S interaction to the EXAFS data, in the event of Cys coordination, deteriorated the fit. Moreover, attempts to fit a [CuI(His)(OH2)]-type multiple scattering model to the EXAFS spectrum did not provide the best overall fit (Table 1). The best fit is obtained using a [CuI(His)2] multiple scattering model, with both Cu− N donor atom distances set to the same value, and with a σ2 of 0.0039 Å2, in agreement with the assessment that Cu(I) coordination environment is highly centrosymmetric (Table 1).

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DISCUSSION It is widely assumed that the divalent oxidation state of Cu(II) is the primary form of Cu in the extracellular environment. This assumption is borne logically from the fact that the potential of the Cu(II)/(I) cycle in complex with amino acids is lower than that of O2 at pH 7.4.58 Thus, in extracellular biological fluids where O2 is present, Cu(I) should be oxidized to Cu(II). 15062

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coordination modes similar to those observed in the cases of Ctr1 or Aβ; specifically, how strongly and with which donor groups does HSA bind to Cu(I)? Our experiments here confirm previous reports that when Cu(II) is bound by HSA, it can be reduced by ascorbate, even under aerobic conditions, and we demonstrate that the extent of Cu(II) reduction depends on the ratio of ascorbate/Cu. This conclusion is reached from both UV−vis data presented in Figure 2, as well as from XAS analysis on samples of Cu(II)HSA complexes exposed to ascorbate, which are clearly in the Cu(I) oxidation state after reaction for 30 min in HEPES buffered solutions at pH 7.4. On the contrary, and consistent with previous reports,36,37 a model peptide of the Cu(II) coordination sequence of HSA is not reduced under the same conditions because it does not possess a Cu(I) binding site. These observations suggest that HSA possesses a Cu(I) binding site, which is yet to be located. The experiments performed here are done at concentrations of the CuHSA complex that are much higher than that of biological fluids due to limits of detection by UV−vis and XAS, but results suggest that the ratio of ascorbate/CuHSA complex may be important in producing the Cu(I)HSA complex. In blood, where the concentration of HSA is approximately 0.6 mM and is fractionally occupied by Cu (approximately 0.05 mM CuHSA), and ascorbate ranges in concentration from 0.2 to 0.8 mM, the ratio of ascorbate/Cu is at least 4. According to our experiments, the Cu(I)HSA complex should represent a significant proportion of Cu bound to HSA in blood where ascorbate concentrations vary, but are at a near steady state. The HSA protein has high 10 fm apparent affinity (log KCu(I):HSA = 14.0 in 50 mM HEPES buffer at pH 7.4) for Cu(I). This affinity is more than 2 orders of magnitude greater than the reported affinity of HSA for Cu(II) determined under similar conditions (log KCu(II):HSA = 11.4 in 100 mM HEPES buffer or 12.0 in the presence of NaCl at pH 7.4). The calculated apparent affinity for the Cu(I)HSA complex reported here is based on the assumption that there is one high affinity binding site for Cu(I) on HSA. It is possible that HSA possesses more than one Cu(I) binding site; however, under the conditions used here, where there is excess HSA and BCA ligand compared to the amount of Cu(I) in solution, we predict that our assumption of a 1:1 complex for Cu(I)HSA is valid. Cu(I) is bound to HSA in a 2-coordinate [CuI(His)2] complex, analogous in structure to the bis-His sites previously characterized for Aβ and the prion protein octarepeat region.50,57 We note that most of the His residues of HSA are accessible on the protein surface; however, few of these are close enough to a second His residue to serve as mutual donor atoms to the same metal without inducing a conformational change. We propose two such candidate locations: His-67 and His-247 which make up the MBS (Figure 1) and His-3 and His9, the former of which is part of the NTS ATCUN sequence and is in a highly mobile region of the protein not often resolved crystallographically. More work is necessary to identify the location of the Cu(I) binding site(s) which we present here. These results demonstrate that Cu(I) may be an accessible and significant species in extracellular fluids. We propose that a Cu(I)HSA complex may play a significant role in extracellular Cu trafficking and particularly in the delivery of Cu to the cellular Cu acquisition protein, Ctr1, which selectively transports Cu(I) through the plasma membrane.35

Article

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.inorgchem.7b02397. Equilibrium model used to determine the apparent affinity of Cu(I) for HSA; near edge spectra of mixtures of Cu(I), BCA; BCA competition for Cu(I) with several sources of HSA; BCA competition for Cu(I) using different sources of Cu(I) (acetonitrile vs ascorbate); EXAFS curve fitting parameters for the linear bis-His (best fit) model to Cu(I)HSA; reaction of Cu(II)-GGH under anaerobic conditions; and lifetime of Cu(I)HSA complex (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

M. Jake Pushie: 0000-0001-7494-5427 Kathryn L. Haas: 0000-0002-8235-5221 Author Contributions

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS All authors gratefully acknowledge financial support from a Research Corporation for Science Advancement Cottrell Scholar Award, which enabled this collaborative research. K.L.H. and M.S. acknowledge additional support from a Marjorie Neuhoff Summer Science Research Communities Grant and the Department of Chemistry and Physics at Saint Mary’s College. E.S. acknowledges a Kosciuszko Foundation Scholarship. M.J.P. is an Associate member of the Canadian Institutes of Health Research-funded program: Training in Health Research Using Synchrotron Techniques (THRUST). Portions of this research were carried out at the Stanford Synchrotron Radiation Lightsource, a Directorate of SLAC National Accelerator Laboratory and an Office of Science User Facility operated for the U.S. Department of Energy Office of Science by Stanford University. The SSRL Structural Molecular Biology Program is supported by the DOE Office of Biological and Environmental Research, and by the National Institutes of Health, National Center for Research Resources, Biomedical Technology Program (P41RR001209). Portions of this research were enabled by support provided by WestGrid (www.westgrid.ca) and Compute Canada Calcul Canada (www.computecanada.ca).

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REFERENCES

(1) Kaplan, J. H.; Maryon, E. B. How Mammalian Cells Acquire Copper: An Essential but Potentially Toxic Metal. Biophys. J. 2016, 110 (1), 7−13. (2) Solomon, E. I.; Heppner, D. E.; Johnston, E. M.; Ginsbach, J. W.; Cirera, J.; Qayyum, M.; Kieber-Emmons, M. T.; Kjaergaard, C. H.; Hadt, R. G.; Tian, L. Copper Active Sites in Biology. Chem. Rev. 2014, 114 (7), 3659−3853. (3) Boal, A. K.; Rosenzweig, A. C. Structural Biology of Copper Trafficking. Chem. Rev. 2009, 109 (10), 4760−4779.

15063

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Article

Inorganic Chemistry

and Engineering of the Major Zinc Binding Site on Human Albumin. J. Biol. Chem. 2009, 284 (34), 23116−23124. (25) Stewart, A. J.; Blindauer, C. A.; Berezenko, S.; Sleep, D.; Sadler, P. J. Interdomain Zinc Site on Human Albumin. Proc. Natl. Acad. Sci. U. S. A. 2003, 100 (7), 3701−3706. (26) Handing, K. B.; Shabalin, I. G.; Kassaar, O.; Khazaipoul, S.; Blindauerd, C. A.; Stewart, A. J.; Chruszcz, M.; Minor, W. Structural Studies of Zinc Carrier Sites in Human and Horse Serum Albumins, March 2 ed.; RCSB PDB, 2016. http://www.rcsb.org/pdb/explore/ explore.do?structureId=5IJF (accessed September 18, 2017). (27) Sogami, M.; Era, S.; Nagaoka, S.; Kuwata, K.; Kida, K.; Miura, K.; Inouye, H.; Suzuki, E.; Hayano, S.; Sawada, S. HPLC-Studies on Nonmercapt-Mercapt Conversion of Human Serum Albumin. Int. J. Pept. Protein Res. 1985, 25 (4), 398−402. (28) Stamler, J. S.; Jaraki, O.; Osborne, J.; Simon, D. I.; Keaney, J.; Vita, J.; Singel, D.; Valeri, C. R.; Loscalzo, J. Nitric Oxide Circulates in Mammalian Plasma Primarily as an S-Nitroso Adduct of Serum Albumin. Proc. Natl. Acad. Sci. U. S. A. 1992, 89 (16), 7674−7677. (29) Sadler, P. J.; Viles, J. H. 1H and 113Cd NMR Investigations of Cd2+ and Zn2+ Binding Sites on Serum Albumin: Competition with Ca2+, Ni2+, Cu2+, and Zn2+. Inorg. Chem. 1996, 35 (15), 4490−4496. (30) Cabras, T.; Patamia, M.; Melino, S.; Inzitari, R.; Messana, I.; Castagnola, M.; Petruzzelli, R. Pro-Oxidant Activity of Histatin 5 Related Cu(II)-Model Peptide Probed by Mass Spectrometry. Biochem. Biophys. Res. Commun. 2007, 358 (1), 277−284. (31) Harford, C.; Sarkar, B. Neuromedin-C Binds Cu(II) and Ni(II) Via the ATCUN Motif - Implicaions for the CNS and Cancer Growth. Biochem. Biophys. Res. Commun. 1995, 209 (3), 877−882. (32) Kolozsi, A.; Jancso, A.; Nagy, N. V.; Gajda, T. N-Terminal Fragment of the Anti-Angiogenic Human Endostatin Binds Copper(II) with Very High Affinity. J. Inorg. Biochem. 2009, 103 (7), 940− 947. (33) Melino, S.; Garlando, L.; Patamia, M.; Paci, M.; Petruzzelli, R. A Metal-Binding Site Is Present in the Amino Terminal Region of the Bioactive Iron Regulator Hepcidin-25. J. Pept. Res. 2005, 66, 65−71. (34) Wiloch, M. Z.; Wawrzyniak, U. E.; Ufnalska, I.; Bonna, A.; Bal, W.; Drew, S. C.; Wroblewski, W. Tuning the Redox Properties of Copper(II) Complexes with Amyloid-Beta Peptides. J. Electrochem. Soc. 2016, 163 (13), G196−G199. (35) Haas, K. L.; Putterman, A. B.; White, D. R.; Thiele, D. J.; Franz, K. J. Model Peptides Provide New Insights into the Role of Histidine Residues as Potential Ligands in Human Cellular Copper Acquisition Via Ctr1. J. Am. Chem. Soc. 2011, 133 (12), 4427−4437. (36) Burke, S. K.; Xu, Y. L.; Margerum, D. W. Cu(II)Gly(2)HisGly Oxidation by H2O2/Ascorbic Acid to the Cu(III) Complex and Its Subsequent Decay to Alkene Peptides. Inorg. Chem. 2003, 42 (19), 5807−5817. (37) Perrone, L.; Mothes, E.; Vignes, M.; Mockel, A.; Figueroa, C.; Miquel, M.-C.; Maddelein, M.-L.; Faller, P. Copper Transfer from CuAbeta to Human Serum Albumin Inhibits Aggregation, Radical Production and Reduces Abeta Toxicity. ChemBioChem 2010, 11 (1), 110−118. (38) Figueroa-Méndez, R.; Rivas-Arancibia, S. Vitamin C in Health and Disease: Its Role in the Metabolism of Cells and Redox State in the Brain. Front. Physiol. 2015, 6, 397. (39) Lotito, S. B.; Frei, B. The Increase in Human Plasma Antioxidant Capacity after Apple Consumption Is Due to the Metabolic Effect of Fructose on Urate, Not Apple-Derived Antioxidant Flavonoids. Free Radical Biol. Med. 2004, 37 (2), 251−258. (40) Tulipani, S.; Romandini, S.; Busco, F.; Bompadre, S.; Mezzetti, B.; Battino, M. Ascorbate, Not Urate, Modulates the Plasma Antioxidant Capacity after Strawberry Intake. Food Chem. 2009, 117 (1), 181−188. (41) Pace, C. N.; Vajdos, F.; Fee, L.; Grimsley, G.; Gray, T. How to Measure and Predict the Molar Absorption Coefficient of a Protein. Protein Sci. 1995, 4 (11), 2411−2423. (42) Kuipers, B. J. H.; Gruppen, H. Prediction of Molar Extinction Coefficients of Proteins and Peptides Using Uv Absorption of the Constituent Amino Acids at 214 nm to Enable Quantitative Reverse

(4) Gaier, E. D.; Eipper, B. A.; Mains, R. E. Copper Signaling in the Mammalian Nervous System: Synaptic Effects. J. Neurosci. Res. 2013, 91 (1), 2−19. (5) Fenton, H. J. H. LXXIII.-Oxidation of Tartaric Acid in Presence of Iron. J. Chem. Soc., Trans. 1894, 65 (0), 899−910. (6) Buckley, W. T.; Vanderpool, R. A. Analytical Variables Affecting Exchangeable Copper Determination in Blood Plasma. BioMetals 2008, 21 (6), 601−612. (7) Neumann, P. Z.; Sass-Kortsak, A. The State of Copper in Human Serum: Evidence for an Amino Acid-Bound Fraction. J. Clin. Invest. 1967, 46 (4), 646−658. (8) Carter, D. C.; Ho, J. X. Structure of Serum Albumin. Adv. Protein Chem. 1994, 45, 153−203. (9) He, X. M.; Carter, D. C. Atomic Structure and Chemistry of Human Serum Albumin. Nature 1992, 358 (6383), 209−215. (10) Bal, W.; Sokolowska, M.; Kurowska, E.; Faller, P. Binding of Transition Metal Ions to Albumin: Sites, Affinities and Rates. Biochim. Biophys. Acta, Gen. Subj. 2013, 1830 (12), 5444−5455. (11) Bhattacharya, A. A.; Grune, T.; Curry, S. Crystallographic Analysis Reveals Common Modes of Binding of Medium and LongChain Fatty Acids to Human Serum Albumin. J. Mol. Biol. 2000, 303 (5), 721−732. (12) Fasano, M.; Curry, S.; Terreno, E.; Galliano, M.; Fanali, G.; Narciso, P.; Notari, S.; Ascenzi, P. The Extraordinary Ligand Binding Properties of Human Serum Albumin. IUBMB Life 2005, 57 (12), 787−796. (13) Yamasaki, K.; Chuang, V. T. G.; Maruyama, T.; Otagiri, M. Albumin−Drug Interaction and Its Clinical Implication. Biochim. Biophys. Acta, Gen. Subj. 2013, 1830 (12), 5435−5443. (14) Petitpas, I.; Petersen, C. E.; Ha, C.-E.; Bhattacharya, A. A.; Zunszain, P. A.; Ghuman, J.; Bhagavan, N. V.; Curry, S. Structural Basis of Albumin-Thyroxine Interactions and Familial Dysalbuminemic Hyperthyroxinemia. Proc. Natl. Acad. Sci. U. S. A. 2003, 100 (11), 6440−6445. (15) Ghuman, J.; Zunszain, P. A.; Petitpas, I.; Bhattacharya, A. A.; Otagiri, M.; Curry, S. Structural Basis of the Drug-Binding Specificity of Human Serum Albumin. J. Mol. Biol. 2005, 353 (1), 38−52. (16) Roberts, J. R.; Xiao, J.; Schliesman, B.; Parsons, D. J.; Shaw, C. F. Kinetics and Mechanism of the Reaction between Serum Albumin and Auranofin (and Its Isopropyl Analogue) in Vitro. Inorg. Chem. 1996, 35 (2), 424−433. (17) Wiglusz, K.; Trynda-Lemiesz, L. Platinum Drugs Binding to Human Serum Albumin: Effect of Non-Steroidal Anti-Inflammatory Drugs. J. Photochem. Photobiol., A 2014, 289, 1−6. (18) Harford, C.; Sarkar, B. Amino Terminal Cu(II)- and Ni(II)Binding (ATCUN) Motif of Proteins and Peptides: Metal Binding, DNA Cleavage, and Other Properties. Acc. Chem. Res. 1997, 30 (3), 123−130. (19) Rózga, M.; Sokołowska, M.; Protas, A.; Bal, W. Human Serum Albumin Coordinates Cu(II) at Its N-Terminal Binding Site with 1 pm Affinity. JBIC, J. Biol. Inorg. Chem. 2007, 12 (6), 913−918. (20) Sokolowska, M.; Krezel, A.; Dyba, M.; Szewczuk, Z.; Bal, W. Short Peptides Are Not Reliable Models of Thermodynamic and Kinetic Properties of the N-Terminal Metal Binding Site in Serum Albumin. Eur. J. Biochem. 2002, 269 (4), 1323−1331. (21) Sokolowska, M.; Wszelaka-Rylik, M.; Poznanski, J.; Bal, W. Spectroscopic and Thermodynamic Determination of Three Distinct Binding Sites for Co(II) Ions in Human Serum Albumin. J. Inorg. Biochem. 2009, 103 (7), 1005−1013. (22) Hureau, C.; Eury, H.; Guillot, R.; Bijani, C.; Sayen, S.; Solari, P. L.; Guillon, E.; Faller, P.; Dorlet, P. X-Ray and Solution Structures of Cu(II) GHK and Cu(II) DAHK Complexes: Influence on Their Redox Properties. Chem. - Eur. J. 2011, 17 (36), 10151−60. (23) Bal, W.; Christodoulou, J.; Sadler, P. J.; Tucker, A. Multi-Metal Binding Site of Serum Albumin. J. Inorg. Biochem. 1998, 70 (1), 33− 39. (24) Blindauer, C. A.; Harvey, I.; Bunyan, K. E.; Stewart, A. J.; Sleep, D.; Harrison, D. J.; Berezenko, S.; Sadler, P. J. Structure, Properties, 15064

DOI: 10.1021/acs.inorgchem.7b02397 Inorg. Chem. 2017, 56, 15057−15065

Article

Inorganic Chemistry Phase High-Performance Liquid Chromatography−Mass Spectrometry Analysis. J. Agric. Food Chem. 2007, 55 (14), 5445−5451. (43) Xiao, Z.; Donnelly, P. S.; Zimmermann, M.; Wedd, A. G. Transfer of Copper between Bis(Thiosemicarbazone) Ligands and Intracellular Copper-Binding Proteins. Insights into Mechanisms of Copper Uptake and Hypoxia Selectivity. Inorg. Chem. 2008, 47 (10), 4338−4347. (44) Chong, L. X.; Ash, M.-R.; Maher, M. J.; Hinds, M. G.; Xiao, Z.; Wedd, A. G. Unprecedented Binding Cooperativity between CuI and CuII in the Copper Resistance Protein CopK from Cupriavidus Metallidurans CH34: Implications from Structural Studies by NMR Spectroscopy and X-Ray Crystallography. J. Am. Chem. Soc. 2009, 131 (10), 3549−3564. (45) Faller, P.; Hureau, C.; Dorlet, P.; Hellwig, P.; Coppel, Y.; Collin, F.; Alies, B. Methods and Techniques to Study the Bioinorganic Chemistry of Metal−Peptide Complexes Linked to Neurodegenerative Diseases. Coord. Chem. Rev. 2012, 256 (19−20), 2381−2396. (46) Williams-Smith, D. L.; Bray, R. C.; Barber, M. J.; Tsopanakis, A. D.; Vincent, S. P. Changes in Apparent pH on Freezing Aqueous Buffer Solutions and Their Relevance to Biochemical ElectronParamagnetic-Resonance Spectroscopy. Biochem. J. 1977, 167 (3), 593−600. (47) Sokolowska, M.; Bal, W. Cu(Ii) Complexation by ″NonCoordinating″ N-2-Hydroxyethylpiperazine-N′-2-Ethanesulfonic Acid (Hepes Buffer). J. Inorg. Biochem. 2005, 99 (8), 1653−1660. (48) Hegetschweiler, K.; Saltman, P. Interaction of Copper(Ii) with N-(2-Hydroxyethyl)Piperazine-N′-Ethanesulfonic Acid (Hepes). Inorg. Chem. 1986, 25 (1), 107−109. (49) Pushie, M. J.; Shaw, K.; Franz, K. J.; Shearer, J.; Haas, K. L. Model Peptide Studies Reveal a Mixed Histidine-Methionine Cu(I) Binding Site at the N-Terminus of Human Copper Transporter 1. Inorg. Chem. 2015, 54, 8544. (50) Pushie, M. J.; Nienaber, K. H.; McDonald, A.; Millhauser, G. L.; George, G. N. Combined EXAFS and DFT Structure Calculations Provide Structural Insights into the 1:1 Multi-Histidine Complexes of Cu-II, Cu-I, and Zn-II with the Tandem Octarepeats of the Mammalian Prion Protein. Chem. - Eur. J. 2014, 20 (31), 9770−9783. (51) McDonald, A.; Pushie, M. J.; Millhauser, G. L.; George, G. N. New Insights into Metal Interactions with the Prion Protein: EXAFS Analysis and Structure Calculations of Copper Binding to a Single Octarepeat from the Prion Protein. J. Phys. Chem. B 2013, 117 (44), 13822−13841. (52) George, G. N.; Pickering, I. J. Exafspak. http://www-ssrl.slac. stanford.edu/exafspak.html. (53) Rehr, J. J.; Mustre de Leon, J.; Zabinsky, S. I.; Albers, R. C. Theoretical X-Ray Absorption Fine-Structure Standards. J. Am. Chem. Soc. 1991, 113 (14), 5135−5140. (54) Lau, S. J.; Kruck, T. P. A.; Sarkar, B. Peptide Molecule Mimicking Copper(II) Transport Site of Human-Serum Albumin Comparative Study between Synthetic Site and Albumin. J. Biol. Chem. 1974, 249 (18), 5878−5884. (55) Bar-Or, D.; Winkler, J. V.; VanBenthuysen, K.; Harris, L.; Lau, E.; Hetzel, F. W. Reduced Albumin-Cobalt Binding with Transient Myocardial Ischemia after Elective Percutaneous Transluminal Coronary Angioplasty: A Preliminary Comparison to Creatine Kinase-Mb, Myoglobin, and Troponin I. Am. Heart J. 2001, 141 (6), 985−991. (56) Kau, L. S.; Spirasolomon, D. J.; Pennerhahn, J. E.; Hodgson, K. O.; Solomon, E. I. X-Ray Absorption Edge Determination of the Oxidation State and Coordination Number of Copper. Application to the Type 3 Site in Rhus Vernicifera Laccase and Its Reaction with Oxygen. J. Am. Chem. Soc. 1987, 109 (21), 6433−6442. (57) Shearer, J.; Szalai, V. A. The Amyloid-Beta Peptide of Alzheimer’s Disease Binds Cu-I in a Linear Bis-His Coordination Environment: Insight into a Possible Neuroprotective Mechanism for the Amyloid-Beta Peptide. J. Am. Chem. Soc. 2008, 130 (52), 17826− 17835.

(58) Youngblood, M. P.; Margerum, D. W. Cyclic Voltammetric Studies of the Reduction of Copper(Ii)-Peptide Complexes. J. Coord. Chem. 1981, 11 (2), 103−110. (59) Rael, L. T.; Bar-Or, R.; Mains, C. W.; Slone, D. S.; Levy, A. S.; Bar-Or, D. Plasma Oxidation-Reduction Potential and Protein Oxidation in Traumatic Brain Injury. J. Neurotrauma 2009, 26 (8), 1203−1211. (60) Schafer, F. Q.; Buettner, G. R. Redox Environment of the Cell as Viewed through the Redox State of the Glutathione Disulfide/ Glutathione Couple. Free Radical Biol. Med. 2001, 30 (11), 1191− 1212. (61) Turell, L.; Radi, R.; Alvarez, B. The Thiol Pool in Human Plasma: The Central Contribution of Albumin to Redox Processes. Free Radical Biol. Med. 2013, 65, 244−253. (62) Grosz, H. J.; Farmer, B. B. Reduction-Oxidation Potential of Blood as a Function of Partial Pressure of Oxygen. Nature 1967, 213 (5077), 717. (63) Himes, R. A.; Park, G. Y.; Barry, A. N.; Blackburn, N. J.; Karlin, K. D. Synthesis and X-Ray Absorption Spectroscopy Structural Studies of Cu(I) Complexes of Histidylhistidine Peptides: The Predominance of Linear 2-Coordinate Geometry. J. Am. Chem. Soc. 2007, 129 (17), 5352−5353. (64) Himes, R. A.; Park, G. Y.; Siluvai, G. S.; Blackburn, N. J.; Karlin, K. D. Structural Studies of Copper(I) Complexes of Amyloid-Beta Peptide Fragments: Formation of Two-Coordinate Bis(Histidine) Complexes. Angew. Chem., Int. Ed. 2008, 47 (47), 9084−9087. (65) Schwab, S.; Shearer, J.; Conklin, S. E.; Alies, B.; Haas, K. L. Sequence Proximity between Cu(II) and Cu(I) Binding Sites of Human Copper Transporter 1 Model Peptides Defines Reactivity with Ascorbate and O2. J. Inorg. Biochem. 2016, 158, 70−76.

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DOI: 10.1021/acs.inorgchem.7b02397 Inorg. Chem. 2017, 56, 15057−15065