Copper Environment in Artificial Metalloproteins Probed by Electron

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The Journal of Physical Chemistry

Monday, July 13, 2015 Revised for Journal of Physical Chemistry B Special Issue (Festschrift) in Honor of Professor Wolfgang Lubitz

The Copper Environment in Artificial Metalloproteins Probed by EPR Spectroscopy Marco Flores, Tien L. Olson, Dong Wang, Selvakumar Edwardraja, Sandip Shinde, JoAnn C. Williams, Giovanna Ghirlanda, and James P. Allen* Department of Chemistry and Biochemistry, Arizona State University, Tempe, Arizona 85287-1604, United States

Keywords: circular dichroism, copper proteins, electron paramagnetic resonance, four-helix bundles, hyperfine coupling, metal-binding peptides

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ABSTRACT: The design of binding sites for divalent metals in artificial proteins is a productive platform for examining the characteristics of metal-ligand interactions. In this report, we investigate the spectroscopic properties of small peptides and four-helix bundles that bind Cu(II). Three small peptides, consisting of fifteen amino acid residues, were designed to have two arms that each contains a metal-binding site comprised of different combinations of imidazole and carboxylate side chains. Two four-helix bundles each had a binding site for a central dinuclear metal cofactor, with one design incorporating additional potential metal ligands at two identical sites. The small peptides displayed pH-dependent, metal-induced changes in the circular dichroism spectra, consistent with large changes in the secondary structure upon metal binding, while the spectra of the four-helix bundles showed a predominant α-helix content but only small structural changes upon metal binding. Electron paramagnetic resonance spectra were measured at X-band revealing classic Cu(II) axial patterns with hyperfine coupling peaks for the small peptides and four-helix bundles exhibiting a range of values that were related to the specific chemical natures of the ligands. The variety of electronic structures allow us to define the distinctive environment of each metal-binding site in these artificial systems, including the designed additional binding sites in one of the four-helix bundles.


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INTRODUCTION Transition metals are present in proteins as redox-active cofactors that are versatile in facilitating many different biological reactions, including those occurring in photosynthesis, respiration, nitrogen fixation, and oxygen metabolism. The functional properties of copper and other metal ions in proteins are strongly influenced by their coordination. These metals are typically coordinated in proteins by amino acid side chains, from residues such as His, Cys, Glu, and Asp, although in some cases the coordinating ligands include backbone atoms, bound water, or non-protein components1,2. Characterization of metal binding to small peptides has contributed to our understanding of affinity and selectivity, for example Margerum and coworkers investigated the binding of copper to histidine-containing tripeptides and tetrapeptides3,4. A rich history exists of using spectroscopic investigations, notably electron paramagnetic resonance (EPR) spectroscopy, to correlate the electronic structure of metal centers in proteins, in particular copper centers, with their chemical properties5. Copper is generally available in the environment and capable of redox activity, and is commonly found in proteins serving in a number of roles, including transferring electrons, transporting molecular oxygen, and facilitating enzymatic reactions such as substrate activation and reduction6. Copper is the only metallic element other than iron that is utilized for long-range electron transfer in cells, with blue copper proteins, whose name derives from the intense blue absorption near 630 nm associated with their mononuclear copper centers, playing critical roles in carrying electrons between cytochrome b6f and photosystem I in photosynthesis and as electron donors for bacterial terminal oxidases in respiration. In nitrogen metabolism, the reduction of nitrite to nitric oxide is driven by the two copper centers in nitrite reductase. Laccases, which contain a trinuclear copper center, are involved in many diverse physiological functions including



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morphogenesis, pathogenesis, and lignin synthesis, through their ability to oxidize a range of aromatic substrates. In addition, copper will bind to the imidazole and carboxylate side chains of proteins and can be used as a general probe of non-copper binding proteins such as photosystem II7,8. The synthesis of peptides that bind copper and other metals makes available an experimental means to investigate features of the binding sites relevant to the properties of complex metal cofactors in natural metalloproteins. In some cases, designs incorporate unstructured peptides that become more ordered after binding metal cofactors9–13. For example, measurements on small peptides provided insight into copper binding to prion proteins, which are associated with a number of transmissible spongiform encephalopathies, and may play a role in neurodegenerative diseases14–17. Both EPR and nuclear magnetic resonance spectroscopies have been applied to define the interactions between prion proteins and copper ions, such as the coordination and binding affinity, so as to elucidate the role of copper in the mechanism of the disease18. The design of a series of small peptides facilitated identification of the binding sites in a repeated eight-residue copper-binding domain found in prion proteins19. In this approach, the sensitivity of EPR spectra to the environment of the metal ion allows detailed investigation of the coordinating atoms contributed by the small metal-binding peptides. The design of novel metal sites in artificial proteins provides an opportunity to engineer model systems that reproduce specific characteristics of larger metalloproteins20–23. For example, peptides based on bacterioferritin include a dinuclear metal-binding site and function as a photosystem II mimic24–26. In some cases the metal cofactors are capable of catalyzing redox reactions, although the rates are generally much slower than observed for natural enzymes27–33. One of the templates for this model-protein approach is the four-helix bundle, as the interface


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between the helices provides a well-defined pocket for cofactor binding34,35. These designs have been successful in the incorporation of metals as dinuclear cofactors, with a focus on iron, manganese, and zinc, but also including cadmium, cobalt, copper, and nickel35–37. The versatility in cofactor binding afforded by the four-helix bundle enables comparison of designs with variations on the metal-binding sites. One of the common features of metalloproteins is their ability to bind different metals.

For

example, the copper cofactor of azurin can be replaced by a wide range of transition metals. Synthetic systems also exhibit this feature, with four-helix bundles being able to bind zinc, manganese, and iron, as well as other metals35,38. For studies on small peptides and four-helix bundles, the choice of metal can either mimic the natural protein or be substituted to examine different characteristics such as the redox potential. In addition, modifications in the amino acid residues at the metal-binding sites provide a test of the dependence of metal binding on the peptide sequence.

The ability of small peptides and four-helix bundles to bind copper suggests

that protein sequences containing possible metal ligands would in general be disposed to bind copper. Incorporation of different metals allows us both to ask questions about factors determining the metal specificity and to take advantage of the properties of particular metals, such as the paramagnetic nature of Cu(II). EPR spectroscopy is well suited for probing the electronic structure of paramagnetic species bound to proteins and peptides, in particular transition metals such as Cu(II) (d9, S = 1/2, I = 3/2). In a magnetic field, the magnetic energy of the unpaired electron of Cu(II) is determined by two factors. One factor is the Zeeman effect, which is a measure of the energetic changes due to the interaction of the unpaired electron spin with the applied magnetic field B0. The other is the



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hyperfine coupling (hfc) due to the interaction of the unpaired electron spin with the nuclear spin of Cu(II). The spin Hamiltonian, H, summarizes the energetic changes due to these interactions: H = βe S﹒g﹒B0 + h S﹒A﹒I

(1)

where g is the electronic g-tensor, βe is the electron Bohr magneton, S is the electron spin operator, A is the hfc tensor, h is Planck’s constant, and I is the nuclear spin operator, which has a value of 3/2 for the most common isotope 63Cu. Copper is typically coordinated in a distorted octahedral geometry with four in-plane ligands that strongly bind the metal and one or two axial ligands that weakly bind the metal6. Due to the axial symmetry, the electronic structure of the unpaired electron can be characterized with a g-tensor having an axial pattern, i.e. gx = gy = g⊥, and gz = g∥ with g∥ > g⊥. Due to its nuclear spin of 3/2, the EPR spectrum of Cu(II) presents a four-line splitting as a consequence of the electron-nuclear hfc interaction that is usually anisotropic and characterized by an axial tensor with components, A∥ and A⊥. Usually for Cu(II), A∥ is much larger than A⊥, thus it is often the case that hyperfine splitting at g⊥ is not resolved whereas the hyperfine splitting at g∥ is observed in the EPR spectrum. Here we report a characterization of the Cu(II) environment in small peptides and four-helix bundles (Fig. 1). In a series of small peptides, the possible coordinating amino acid residues were systematically altered. The starting point for the design was a published peptide of 15 amino acid residues containing two metal-coordinating sites, with each site having three His residues that could serve as metal ligands39. For two of the peptides investigated, the three His residues were altered to include one or two carboxylates in each binding site. In addition, all three His were changed to Asn, whose side chains would not be expected to coordinate a metal ion. In the second set of metalloproteins, the polypeptide chains were larger, consisting of 52 amino acid residues,


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and designed to dimerize in solution and form four-helix bundles. These designs are based upon the DF2t protein of the Due Ferri (DF), or diiron, family of de novo proteins from DeGrado and coworkers40,41. One of these proteins, designated P0, contains one dinuclear metal site, while a second protein, P1, should be capable of binding additional metals through changes that add potential ligands. The binding of Cu(II) to this collection of small peptides and four-helix bundles was characterized using circular dichroism (CD) and absorption spectroscopy. The electronic structure of the bound metal ions was probed using EPR spectroscopy, allowing comparison of the copper environment in each artificial metalloprotein. MATERIALS AND METHODS Small Peptide Design. Each of the small peptides contains a total of fifteen amino acid residues (Fig. 1). Two identical regions containing amino acid residues for metal binding were symmetrically placed with a central Gly-Pro pair of residues to provide a potential β-turn between the two metal-binding regions. Each peptide also contained a N-terminal Trp residue that allowed monitoring of the peptide concentration using absorption spectroscopy. The designs were based upon a published peptide, identified here as the HHH peptide, that has the sequence WGHGHGHGPGHGHGH39. For the HHH peptide, each metal-binding region has three His residues serving as potential ligands with Gly residues as spacers. Two peptides were designed with substitutions of different combinations of Asp and His residues. The HDH peptide has the sequence WGHGDGHGPGHGDGH in which the central His ligand of each metal-binding site is substituted with Asp. The DHD peptide has the sequence WGDGHGDGPGDGHGD in which two of the His residues in each metal-binding region are substituted with Asp. Thus, the HHH, HDH and DHD peptides all had two potential metal-binding regions consisting of different



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combinations of imidazole and carboxylate side chains. Finally, the NNN peptide has the sequence WGNGNGNGPGNGNGN in which all of the His residues are replaced with Asn. Small Peptide Preparation and Purification. Peptides were synthesized using an automated peptide synthesizer (CEM, North Carolina, USA) on a H-Rink Amide ChemMatrix resin (PCAS-BioMatrix, Québec, Canada), which after cleavage gave a C-terminal amide, or using a Novabiochem Fmoc-Asn(Trt) resin (EMD Chemicals, New Jersey, USA) to yield a C-terminal carboxylate group42. Racemization of the histidine side chain during coupling was reduced by having the microwave turned off and increasing the reaction time. For the synthesis of the HDH and DHD peptides, piperazine was utilized instead of piperidine to limit aspartimide formation. For all peptides, after cleavage from the resin and simultaneous side-chain deprotection by trifluoroacetic acid, the sample was purified through reverse phase high performance liquid chromatography on a HPLC 1525 system (Waters, Massachusetts, USA) using a C18 semipreparative column (Vydac, Illinois, USA) or a C18 analytical column (AAPPTee, Kentucky, USA). For each peptide, the molecular mass was verified by matrix-assisted laser desorption and ionization: time-of-flight mass spectrometry using a DE-STR mass spectrometer (Applied Biosystems, Massachusetts, USA). The instrument was operated under positive ion mode with an α-cyano-4-hydroxycinnamic acid matrix. The purity of each peptide was verified by C18 analytical HPLC. After purification, the peptides were lyophilized and stored at 4 °C. For CD spectroscopy, the HHH, HDH, and DHD peptide concentrations were typically poised at 0.2 mM, and CuSO4 at a 2:1 or 4:1 Cu:peptide molar ratio was added immediately prior to measurement. For measurements at different pH values, the following buffers were used: acetate from pH 3.5-5.5, MES from pH 5.5-6.8, MOPS from pH 6.5-8.0, TAPS from pH 7.8-9.0, and CHES from pH 8.8-10.3, with the pH values adjusted as needed using potassium hydroxide.


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Typically, a 50 mM buffer concentration was used for measurements in the 210-260 nm and 300-700 nm ranges although an 8 mM buffer concentration was used to extend the spectral range to 200-260 nm. For EPR spectroscopy, samples with a peptide concentration of 1 mM were prepared in 50 mM CHES buffer at pH 9.2 with 15% ethylene glycol as a cryoprotectant. Also measured was CuSO4 at a 1 mM concentration in 15% ethylene glycol. To minimize the effect of free Cu(II), a 2:1 molar ratio of metal to peptide was used for the HHH and DHD peptides, and a 1:1 molar ratio was used for the HDH peptide. Four-Helix Bundle Preparation. The P0 and P1 proteins are variants of the DF type of four-helix bundles35. The P0 protein has the same amino acid sequence as the DF2t protein40,41, except for substitution of Gly for Met at the N-terminus and lack of Gly at the C-terminus, and contains metal ligands at Glu 11, Glu 41 and His 44 (Fig. 1). Compared to the P0 protein, the P1 protein has changes, Leu 4 to Glu, Tyr 18 to Glu, Leu 34 to Glu, Ile 37 to His, Leu 48 to Glu and Ile 51 to His, that potentially provide additional Glu and His ligands on either side of the central metal-binding site (Fig. 1). The design and characterization of the P0 and P1 proteins has been reported elsewhere43. Briefly, the P0 and P1 proteins were expressed in Escherichia coli and purified through affinity chromatography using a histidine tag at the N-terminus that was subsequently removed by protease cleavage. In general, the isolated P0 and P1 proteins were dialyzed against 5 mM MOPS, pH 7.6 or water. For metal binding, the P0 and P1 proteins were adjusted to a concentration of 0.01 mM with respect to the monomer in 5 mM MOPS, pH 7.6. After addition of 0.03 mM CuSO4, the protein solutions were rocked at 4° C for 2 hours and then concentrated as necessary using Amicon Ultra-15 3K centrifugal filters (EMD Millipore, Massachusetts, USA). For the CD spectra the proteins were poised at 0.04 mM. The EPR spectra were measured on samples at concentrations



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of 0.15 mM for the P0 protein and 0.32 mM for the P1 protein. Absorption spectra were also measured with CuSO4 added in various ratios to a 0.3 mM P0 sample and a 0.1 mM P1 sample. CD and Absorption Spectroscopy. The CD spectra were recorded on a J-710 spectropolarimeter (JASCO, Tokyo, Japan) with the samples under the flow of nitrogen gas. The samples were placed in 0.1 cm and 1 cm pathlength quartz cuvettes for measurements in the 200-260 nm and 300-700 nm ranges, respectively. Absorption spectra were measured using a Cary 6000i UV-Vis-NIR spectrophotometer (Agilent Technologies, California, USA). EPR Spectroscopy. Studies were performed at the EPR Facility of Arizona State University. Continuous wave (CW) EPR spectra were recorded using an ELEXSYS E580 CW X-band spectrometer (Bruker, Rheinstetten, Germany) equipped with a Model 900 EPL liquid helium cryostat (Oxford Instruments, Oxfordshire, UK). For the measurements of copper bound to the peptides, the magnetic field modulation frequency was 100 kHz, the amplitude was 1 mT, the microwave power was 16 µW, the microwave frequency was 9.43 GHz, the sweep time was 84 s, and the temperature was 30 K. For the four-helix bundles, the experimental spectra were measured using a microwave power of 64 µW at a temperature of 15 K. The EPR spectra were simulated using EasySpin (version 4.5.5), a computational package developed by Stoll and Schweiger44 and based on Matlab (The MathWorks, Massachusetts, USA).

For the small

peptides and the P0 protein, the model used for the EPR simulations considered a single Cu(II) ion (S = 1/2, I = 3/2). simulations.

For the P1 protein, two non-equivalent Cu(II) ions were included in the

The fitting parameters were the g-values (gx, gy and gz), the line widths (∆Bx, ∆By

and ∆Bz), and the hfc splittings (Ax, Ay and Az).

The fitting procedure was similar to the one

previously described by Flores and coworkers45.


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RESULTS Binding of Cu(II) to the Small Peptides Measured using CD Spectroscopy. The binding of Cu(II) to the small peptides was characterized using CD spectroscopy at room temperature in the ultraviolet region from 200 to 260 nm and the visible region from 300 to 700 nm. The signals in the UV region are indicative of the secondary structure of the peptide, which are useful in detecting structural changes of the peptide due to metal binding, and the spectral features in the visible region are due to metal binding and reflect interactions between the protein ligands and metal ions. In the absence of Cu(II), the CD spectrum of the DHD peptide showed features typical of a random coil, namely a minimum at 200 nm and a broad band at 224 nm (Fig. 2). In the presence of Cu(II), no changes to the spectrum were observed at low pH but at high pH the CD spectrum gained new distinctive features, specifically the appearance of a positive band centered at 215 nm in the UV region and two broad bands centered at 327 and 589 nm in the visible region. For the HDH peptide, the presence of Cu(II) resulted in a spectral profile similar to that of the DHD peptide, with a negative band below 205 nm and positive band at 214 nm in the UV region, and positive bands at 325 and 564 nm in the visible region. The CD spectrum for the HHH peptide, with peaks at 348 and 570 nm, is similar to that previously published39. For the NNN peptide, no changes in the CD signal were observed in the presence of Cu(II). When the Cu(II) concentration was systematically increased, the amplitude of the CD spectra reached a maximum value for two Cu(II) per peptide for the HHH, HDH and DHD peptides. The CD spectra of the three peptides showed a similar pH dependence, with larger amplitudes resulting from increasing the pH above pH 5 until the pH value was above 8 (Fig. S1). The CD spectra indicate that the HHH and HDH peptides also bind Ni(II) and the HHH peptide binds Co(II) (Fig. S2).



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Binding of Cu(II) to the Four-Helix Bundles. CD spectra were measured for the P0 and P1 four-helix bundles at 4° C (Fig. 3). The apo-form of the proteins had spectra characteristic of α-helices, demonstrating the presence of a well-defined secondary structure in the absence of the metal. For the apo-P0 protein, the spectra showed a maximum near 190 nm, and minima at 207 and 221 nm. In the presence of Cu(II), the spectral minima were slightly shifted to 208 and 223 nm with minor changes in the relative amplitudes. The spectra of the apo-P1 protein and Cu(II)-bound P1 protein were the same within error, with a peak maximum near 190 nm, and minima at 206 and 222 nm. These minor or negligible shifts of the spectra of the P0 and P1 proteins upon Cu(II) binding indicate that metal binding makes little change in the helical structure. The CD spectra for the P0 and P1 proteins and the small shifts upon Cu(II) binding are similar to those reported for the DF four-helix bundles in the absence and presence of Mn(II), Co(II) and Zn(II)37,46. Absorption measurements showed a broad peak near 650 nm for the P0 protein and near 630 nm for the P1 protein when the proteins were in the presence of Cu(II). Since no absorbance was evident for the apo-protein, these absorption bands are assigned as arising from transitions associated with the bound Cu(II). The amplitudes of the absorption bands increased with addition of Cu(II) until a Cu:protein-monomer ratio of approximately 1:1 for the P0 protein and 3:1 for the P1 protein, corresponding to one and three dinuclear Cu clusters binding to the P0 and P1 four-helix bundles, respectively. Electronic Structure of the Bound Cu(II) Determined using EPR. The EPR measurements of the Cu(II)-bound small peptides were performed using a X-band spectrometer (Fig. 4). The EPR spectra are consistent with a single type II Cu(II) (d9, S = 1/2, I = 3/2) system with four distinctive hfc peaks. Fits using the corresponding spin Hamiltonian are in excellent 12 Environment ACS Paragon Plus

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agreement with the data (Eq. 1).

For each of the three peptides, the spectrum of the bound Cu(II)

showed axial symmetry with g∥-values of 2.213, 2.227, and 2.236 and g⊥-values of 2.061, 2.057, and 2.057 for the HHH, HDH, and DHD peptides, respectively (Table 1). These EPR spectra of Cu(II) bound to the small peptides showed features that are distinct from those present in the spectrum of Cu(II) in solution, demonstrating that such spectral differences are due to the interactions between the peptides and Cu(II). The EPR spectra of Cu(II) bound to the P0 and P1 four-helix bundles had well-defined features (Fig. 5). The spectrum of the P0 protein showed the bound metal to have axial symmetry with four hfc peaks as found for the small peptides. These data were well described using a g∥-value of 2.242, a g⊥-value of 2.059, and hfc values of 5.9 × 10-4, 19.2 × 10-4, and 187.5 × 10-4 cm-1 for Ax, Ay, and A∥, respectively (Table 1). In contrast, the spectrum of the P1 protein has both additional and broader hfc peaks. This spectrum could not be adequately described using parameters associated with one Cu(II) center but were well fitted by assuming that the spectrum had contributions of two Cu(II) centers, which are quite distinctive, for example, the g∥-values are 2.280 and 2.230, and the A∥ values are 177.4 × 10-4 and 193.6 × 10-4 cm-1, for component 1 and 2, respectively. The relative amplitude of component 1 to component 2 is approximately 2:1. DISCUSSION Despite considerable research, detailed mechanisms remain elusive for some fundamental biological processes, such as molecular hydrogen oxidation, nitrogen fixation, and water oxidation, which are facilitated by the metalloproteins hydrogenase, nitrogenase, and photosystem II, respectively. The ability to synthesize metal-binding peptides and proteins provides an avenue for investigating the properties of metal cofactors in these and other natural metalloproteins.



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Small peptides represent a basic, amenable system, while the larger artificial proteins, such as the four-helix bundles, model the complexities of natural proteins, including well-defined secondary structure and folds. The use of copper has the advantages of it being a redox-active metal that readily binds to carboxylate and imidazole side chains in proteins. In addition, the electronic structure of copper is strongly dependent on the geometry of the coordination, allowing the influence of the local protein environment to be accessed using EPR spectroscopy6. In this study, three small peptides were designed with different combinations of imidazole and carboxylate ligands, and two four-helix bundle proteins were designed with different numbers of binding sites that also contain imidazole and carboxylate ligands (Fig. 1). For the small HHH, HDH, and DHD peptides, the binding of Cu(II) resulted in large changes in the secondary structure as monitored by CD spectral changes in the UV region, with the binding being strongly pH dependent (Fig. 2). The CD bands observed in the visible region arise from three overlapping d-d transitions that are sensitive to the specific coordination geometry of the copper, the chirality of the adjoining amino acid residues, and temperature, thus yielding a complex set of factors that influence the shape and position of this peak47,48. Different environments of the metal-binding site in these three peptides are evident in the differences of the CD spectra in the visible region (Fig. 2). The P0 and P1 four-helix bundles have marked differences in their CD spectra compared to those for the three small peptides (Fig. 3). The CD spectra show a pronounced α-helical character that is present in the apo-state with only minor alterations due to the binding of the copper (Fig. 3). Thus, the binding of copper results in a large conformation change for the small peptides but not the four-helix bundles, consistent with the behavior expected for these designs and observed previously for the HHH peptide and DF proteins37,39,46.


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For all of the small peptides and four-helix bundles, the bound copper has a characteristic type II Cu(II) EPR spectrum, with each one having distinctive features due to the influence of the particular nature of the coordination geometry on the electronic structure (Figs. 4, 5, Table 1). The observed differences in the EPR spectra reflect specific axial and equatorial features for the copper centers in the small peptides and four-helix bundles and can be understood in terms of Jahn-Teller distortion associated with the bound copper. According to their unique spectroscopic features, mononuclear copper cofactors are divided into different categories6. Mononuclear copper centers are usually divided into type I or type II centers. The type I centers of copper proteins, such as azurin, typically have the copper ion coordinated by two His and a Cys, defining a trigonal plane around the copper, with an additional axial ligand from Met in some cases. The type II centers are found many copper proteins, such as nitrite reductase, and typically have a square-planar or tetrahedrally distorted square-planar coordination formed by His, Tyr, Asp, or H2O ligands. In addition, there are several other classifications of copper centers. For example, the CuA centers are binuclear with the copper ions coordinated by two His and bridged by two Cys, as found in cytochrome c oxidase, with the mixed valence nature of the center producing EPR spectra with seven lines. Each type of copper center has specific features in the EPR spectrum and hence characteristic EPR parameters. Type I copper usually has a small A∥ (< 90 × 10-4 cm-1), while a type II copper center typically has a large A∥ (> 140 × 10-4 cm-1). Since A∥ is usually much larger than A⊥, the hyperfine splitting is observed at g∥ but not g⊥ in the EPR spectra49. In comparing the measured spectra with these profiles for the different types of copper centers, we observe that the fitted A∥ values of the spectra are all greater than 140 × 10-4 cm-1, ranging from 173 × 10-4 to 194 × 10-4 cm-1, and so the copper centers of the small peptides and four-helix bundles are all assigned as type II.



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The differences in the EPR parameters obtained for the small peptides, and hence the electronic structures of the bound Cu(II), suggest a relationship between the binding characteristics and the chemical nature of the ligands.

The g-tensor represents a measure of the

integral properties of Cu(II) and its ligands49. The g-tensor obtained for Cu(II) in solution, as expected, has axial symmetry with values that are typical for Cu(II) in an octahedral geometry (Table 1). In addition, the electron-nuclear hfc interaction provides information about local properties of the Cu(II) complex, i.e. the electron spin density distribution.

In particular, the

parameter A∥ represents an indirect measure of the electron spin density at the Cu(II) nucleus. The values of g∥ and A∥ obtained for Cu(II) in solution indicate that the metal is coordinated to six ligands with at least four being oxygen atoms (Table 1). When Cu(II) was bound to the peptides, the g-values and the value of A∥ changed. For the g-values, this is most clearly seen from the axial distortion parameter, which had values of 16.1% for Cu(II) in H2O and 7.4%, 8.3%, and 8.7% for Cu(II) bound to the HHH, HDH, and DHD peptides, respectively. The decrease of the axial distortion parameter upon peptide binding is in line with a change in the coordination geometry of Cu(II).

Furthermore, the increase of the value of A∥ from 134.1 × 10-4 cm-1 (H2O)

to 191.6 × 10-4 cm-1, 190.0 × 10-4 cm-1, and 172.7 × 10-4 cm-1 for the HHH, HDH, and DHD peptides, respectively, indicates a decrease in the number of ligands upon peptide binding. Both the g-tensor and hfc parameters suggest that Cu(II) binds to the small peptides in a distorted square planar configuration with less than six ligands. For the small peptides, the bound Cu(II) could be coordinated to nitrogen or oxygen atoms from the side chains, backbone, or bound water molecules. The HHH peptide showed the smallest g∥ (2.213) and the largest A∥ (191.6 × 10-4 cm-1).

These findings indicate that Cu(II) is

coordinated to the HHH peptide via four nitrogens in a close square planar geometry as


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previously reported39.

Presumably, three of the nitrogens are from the imidazole side chains

and the fourth is provided by the backbone.

For the HDH peptide, in which the central His

ligand (nitrogen donor) of each metal-binding site was substituted with Asp (oxygen donor), the value of g∥ increased to 2.227 and the value of A∥ decreased to 190.0 × 10-4 cm-1.

When two of

the His residues in each metal-binding region were substituted with Asp in the DHD peptide, the impact on g∥ (2.236) and A∥ (172.7 × 10-4 cm-1) was even larger. This trend indicates a change in the nature of the ligands of Cu(II), i.e. a change from purely nitrogen ligands towards mixed nitrogen and oxygen ligands.

According to the phenomenological model proposed by Peisach

and Blumberg49, the values of g∥ and A∥ determined for the HDH and DHD peptides suggest that Cu(II) is bound to either one oxygen and three nitrogen ligands or two nitrogen and two oxygen ligands. The observed correlation between the EPR spectra of the three small peptides and their copper coordination provides the opportunity to use the small peptides as a model system for assigning features of EPR spectra associated with copper-binding sites in proteins. For the P0 four-helix bundle, the EPR spectrum was well described by parameters that are similar to those of the DHD peptide (Table 1). For example, the values of g∥ and the axial distortion are 2.242 and 8.9%, respectively, for the P0 protein, and 2.236 and 8.7%, respectively, for the DHD peptide. The binding of copper to one imidazole and two carboxylates is consistent with the design of this metalloprotein (Fig. 1). While there is no three-dimensional structure of a copper-bound four-helix bundle, structures have been determined for DF proteins with Cd(II), Co(II), Mn(II), and Zn(II)35. These structures show that the metals are dinuclear centers bound at the designed site, with each metal ion being five-coordinated by three carboxylates and one histidine.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

The EPR spectrum of the P1 four-helix bundle is best described as arising from two Cu(II) sites. For component 2, the fitted parameters are very close to the parameters for the P0 protein, for example, the g∥, A∥, and axial distortion values are 2.230, 193.6 × 10-4 cm-1, and 7.3% for component 2 compared to values of 2.242, 187.5 × 10-4 cm-1, and 8.9% for the P0 protein. The similarity of the spectral parameters between P0 and component 2 suggests that this copper-binding site in the P1 protein corresponds to the central copper-binding site in the P0 protein. The second set of spectral features observed for the P1 four-helix bundle are described by the parameters of component 1. The fitted parameters for component 1 of 2.280 for g∥ and 177.4 × 10-4 cm-1 for A∥ are much different than the corresponding parameters of 2.230 for g∥ and 193.6 × 10-4 cm-1 for component 2, and the axial distortion is much larger with a value of 10.8% compared to 7.3%, indicating a more open environment for the Cu(II) center associated with component 1. The nearly 2:1 ratio for the relative amplitudes of component 1 and 2 obtained from the fit indicates that component 1, with twice the contribution to the EPR spectrum, is associated with two indistinguishable binding sites. Component 1 is then assigned to the additional copper-binding sites designed in the P1 protein. The fits of the EPR spectra are consistent with the P1 protein containing three binding sites, one that is similar to the site in the P0 protein, and two that are identical to each other but distinct from the site in the P0 protein. This interpretation is corroborated by the observation that the amplitude of the visible absorption peak reached a maximum for a 3:1 Cu:P1-monomer ratio (or equivalently three dinuclear Cu(II) sites in each four-helix bundle). Thus the data reveal three metal-binding sites in the P1 protein compared to one metal-binding site in the P0 protein, in agreement with the design of a central dinuclear metal-binding site common to both the P0 and P1 proteins, with two additional


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symmetrically-related dinuclear metal-binding sites closer to the ends of the helices in the P1 protein (Fig. 1). The ability to bind different metals is evident for the small peptides as all three peptides bind Cu(II), while the HHH and HDH peptides also bind Ni(II), and the HHH peptide binds Co(II). This dependence of metal binding upon the peptide sequence is consistent with the observation that the binding of metal ions to proteins is determined primarily by the structural arrangement of side chains that serve as coordinating ligands, with other factors, such as protein flexibility, electrostatic interactions, and steric considerations, having secondary contributions1,2,50.

For

divalent transition metals, the Irving-Williams series provides a relationship between the relative stability of divalent metals for a given set of ligands and the properties of the metals themselves, notably the ligand field stabilization energies51,52. According to this series, the order of increasing ligand field stabilization energy is Mn(II) < Fe(II) < Co(II) < Ni(II) < Cu(II) > Zn(II), correlating with the observation that Cu(II) binds to all three of the small peptide designs.

This

series is consistent with a general utility of using Cu(II) to probe metal-binding sites in proteins. CONCLUSIONS The ability of small peptides and four-helix bundles to bind Cu(II) has been examined using optical and EPR spectroscopies. The circular dichroism spectra show large changes in conformation associated with metal binding for the small peptides, while the four-helix bundles exhibited only small changes. The EPR spectroscopic signatures reveal these metal centers to all have type II Cu(II) electronic structures, with each peptide having different g-values and hfc parameters. The series of small peptides provided a well-defined test system for understanding the properties of the bound copper, as comparison of the spectra showed the effects of systematically varying the number of imidazole and carboxylate ligands on the metal binding and



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coordination. For the two four-helix bundles, the imidazole and carboxylate proportions in the ligands are maintained, but the location of the binding sites differ, thus altering the broader protein surroundings and yielding the two distinctive environments revealed in the EPR spectra. These results demonstrate the utility of EPR measurements on these classic type II Cu(II) centers for interpretation of design alterations in terms of the electronic structure and nature of the geometry of the metal-binding sites. AUTHOR INFORMATION Corresponding Author *E-mail: [email protected]. Tel: 480-965-8241. Fax: 480-965-2747. Funding Sources This material is based upon work supported by the National Science Foundation, CHE 1505874, and as part of the Center for Bio-Inspired Solar Fuel Production, an Energy Frontier Research Center funded by the U.S. Department of Energy, Office of Science, Office of Basic Energy Sciences under Award Number DE-SC0001016. ACKNOWLEDGMENTS We thank E. Canarie, C. Helterbran, and A. Hauptli for assistance with preparation of the proteins. SUPPORTING INFORMATION Detailed description and figures of CD spectra showing the pH dependence and the binding of different divalent metals to the small peptides are provided. This material is available free of charge via the Internet at http://pubs.acs.org. ABBREVIATIONS


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CD, circular dichroism; DF, Due Ferri; EPR, electron paramagnetic resonance; hfc, hyperfine coupling REFERENCES (1)

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Table 1. Summary of parameters obtained from the fits of X-band EPR spectra of Cu(II) in solution and bound to the small peptides and four-helix bundles. Parametersa

H2O

gx

2.082

2.045

2.049

gy

2.090

2.076

g∥

2.421

g⊥b

P0

P1 #1

P1 #2

2.043

2.051

2.055

2.049

2.065

2.071

2.067

2.061

2.106

2.213

2.227

2.236

2.242

2.280

2.230

2.086

2.061

2.057

2.057

2.059

2.058

2.078

16.1

7.4

8.3

8.7

8.9

10.8

7.3

∆Bx (MHz)

133.7

132.2

157.6

134.3

103.4

126.2

95.4

∆By (MHz)

208.5

338.1

314.1

367.4

251.0

346.4

312.2

∆Bz (MHz)

83.3

278.2

245.8

200.4

239.1

289.1

205.4

Ax(Cu) (10-4 cm-1)

0.0

3.0

3.2

6.9

5.9

3.2

6.1

Ay(Cu) (10-4 cm-1)

13.1

8.6

20.3

22.9

19.2

25.2

39.0

A∥(Cu) (10-4 cm-1)

134.1

191.6

190.0

172.7

187.5

177.4

193.6

Axial distortionc (%)

a

HHH

HDH

DHD

The fitting parameters were the three g-values, gx, gy, and g∥, where g∥ is defined as gz, the three

line widths, ∆Bx, ∆By, and ∆Bz, and the three hfc constants, Ax, Ay, and A∥, where A∥ is defined as Az. Errors for gx, gy, g∥, and g⊥ are ±0.001. respectively.

Errors for ∆Bx, ∆By, and ∆Bz are ±5, ±30, and ±20 MHz,

Errors for Ax, Ay, and A∥ are ±2 × 10-4, ±4 × 10-4, and ±1 × 10-4 cm-1, respectively.

b

g⊥ was calculated as [(gx2 + gy2) / 2]1/2.

c

The axial distortion parameter is defined as [(g∥ − g⊥)/g⊥] x 100%.


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FIGURE CAPTIONS Figure 1. Schematic view of the sequences of the small peptides (top) and four-helix bundles (bottom) with bound copper ions. The HHH, HDH, and DHD peptides each contain fifteen amino acid residues and two mononuclear metal-binding sites. The HHH peptide has His at positions 3, 5, 7, 11, 13, and 15. The HDH peptide has His at positions 3, 7, 11, and 15, and Asp at 5 and 13. The DHD peptide has His at positions 5 and 13 and Asp at 3, 7, 11, and 15. The P0 protein forms a four-helix bundle consisting of an antiparallel dimer with 52 amino acid residues in each monomer and ligands for a dinuclear metal-binding site involving Glu 11, Glu 41 and His 44. The P1 protein has changes that introduce Glu at positions 4, 18, 34, and 48, and His at positions 37 and 51, potentially adding two new dinuclear metal-binding sites. Figure 2. CD spectra of the small peptides. (a) CD spectra for the DHD peptide in the absence of Cu(II) at pH 3.6 (apo, black) and with Cu(II) at pH values of 3.6 (dark red) and 8.0 (red). (b) CD spectra for the HHH (blue), HDH (green), DHD (red), and NNN (black) peptides in the presence of Cu(II) at pH 8. Figure 3. CD and absorption spectra of the four-helix bundles. (a) CD spectra for the P0 (blue) and P1 (red) proteins with Cu(II) compared to the absence of Cu(II) (apo, black). (b) Absorption spectra for the P0 (blue) and P1 (red) proteins with Cu(II). Figure 4. EPR spectra of Cu(II) in H2O and bound to the small peptides measured at X-band (9.4 GHz) and a temperature of 30 K. Also shown are best fits of the data (dotted lines) using Eq. 1 with parameters summarized in Table 1. Figure 5. EPR spectra of Cu(II) bound to the four-helix bundles measured at X-band (9.4 GHz) and a temperature of 15 K. The data (solid lines) are shown with the best fits using Eq. 1 (dotted



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lines). For the P1 protein, the spectral contributions of the two components are shown separately (dashed lines). All parameters are summarized in Table 1.


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1 W


G

15

1 2 HD HD 3 G G 4 5 HD HD G HD G P G HD G 6 7 8 9 1 11 10 11 G D Y LE R E L Y K L E Q Q A M K L YE R E A S E K A R 12 13 44 41 52 N 14 P N IH T E LE W E I H K E E D E L IH K Q LE V S K K E 15 16 17 18 19 20 21 E K K S V LE Q K IH L E D E E K H I E W LE E T IH N 22 P N 23 41 44 52 24R Paragon A K E S A E R Y E L ACS K M A QPlusQEnvironment E L K Y L E R LE Y D G 25 26 1 11 27


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Figure 2 133x215mm (300 x 300 DPI)

ACS Paragon Plus Environment

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Figure 3 133x215mm (300 x 300 DPI)



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Figure 4 241x324mm (300 x 300 DPI)


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Figure 5 232x286mm (300 x 300 DPI)



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Table of Contents Figure 39x31mm (300 x 300 DPI)


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Copper Environment in Artificial Metalloproteins Probed by Electron

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