The affinity of HGGG, GHGG, GGHG, and GGGH peptides for copper (II) and the structures of their complexes An ab initio study1

See discussions, stats, and author profiles for this publication at: https://www.researchgate.net/publication/237152620 The affinity of HGGG, GHGG, GGHG, and GGGH peptides for copper(ii) and the structures of their complexes - An ab initio... Article in Canadian Journal of Chemistry · June 2009 DOI: 10.1139/V09-034 CITATIONS READS 9 312 4 authors, including: M. Jake Pushie Arvi Rauk 32 PUBLICATIONS 461 CITATIONS 279 PUBLICATIONS 9,194 CITATIONS University of Saskatchewan SEE PROFILE All content following this page was uploaded by Arvi Rauk on 28 December 2016. The user has requested enhancement of the downloaded file. The University of Calgary SEE PROFILE 942 The affinity of HGGG, GHGG, GGHG, and GGGH peptides for copper(II) and the structures of their complexes — An ab initio study1 Stephen D. Barry, Gail A. Rickard, M. Jake Pushie, and Arvi Rauk Abstract: The structures and relative free energies in aqueous solution of the Cu(II) complexes of the ‘‘histidine walk’’ peptides, AcHGGGNH2, AcGHGGNH2, AcGGHGNH2, and AcGGGHNH2, were determined as a function of pH. Numerous structures of each species were found by gaseous- and solution-phase geometry optimization at the B3LYP/6–31G(d) level, and the effect of solvation estimated by the IEFPCM continuum solvation model. Free energies of solvation of the ionic species are large and favour structures with an extended peptide chain. In all Cu(II)–peptide complexes, deprotonation of two amide groups occurs readily at or below pH 7. In each system, the most abundant species at pH 7 is a neutral 1:1 complex with N3O1 coordination pattern. Binding in the forward direction toward the C terminus is preferred. The results are compared to recent experimental spectroscopic and potentiometric studies on related systems. Alternative explanations are offered for some of the experimental observations. Key words: copper–peptides, theoretical, DFT, prion, binding affinities, CD spectra. Résumé : On a déterminé les structures et les énergies libres, en solutions aqueuses et en fonction du pH, des complexes de Cu(II) des peptides de la série histidine, AcHGGGNH2, ACGHGGNH2, AcGGHGNH2 et ACGGGHNH2. On a déterminé les diverses structures de chacune des espèces, en phase gazeuse et en solution, par une optimisation de la géométrie au niveau B3LYP/6-31G(d) et on a évalué l’effet de solvatation par le modèle de solvatation continue IEEPCM. Les énergies libres de solvatation des espèces ioniques sont importantes et elles favorisent les structures avec une chaı̂ne peptidique étendue. Dans tous les complexes Cu(II)–peptide, la déprotonation de deux groupes amides se produit facilement à des pH égal ou inférieurs à 7. Dans chaque système, l’espèce la plus abondante au pH 7 est un complexe 1:1 neutre comportant un patron de coordination N3O1. La fixation est favorisée dans la direction allant vers la position C-terminale. On compare les résultats obtenus ici avec ceux rapportés récemment pour des études expérimentales spectroscopiques et potentiométriques sur des systèmes apparentés. Des explications alternatives sont proposées pour un certain nombre d’observations expérimentales. Mots-clés : complexes cuivre–peptides, théorique, théorie de la fonctionnelle de densité (TFD), affinités de fixation, spectres de dichroı̈sme circulaire (DC). [Traduit par la Rédaction] Introduction The pathology of many neurodegenerative diseases involves the disruption of normal trafficking of a metal ion which is ultimately related to its binding to a biologically prevalent protein or peptide. These include Alzheimer’s, Parkinson’s, and Huntington’s diseases, as well as the prion diseases, also known as transmissible spongiform encephalopathies (TSEs), which result in the deterioration of neurons.2,3 The misfolding of these proteins/peptides, which in some instances may involve metal binding, is believed to cause the observed neurotoxicity of these species.2,4,5 Especially in the case of TSEs and Alzheimer’s disease (AD), copper is involved in peptide–protein binding, and has been Received 11 November 2008. Accepted 21 January 2009. Published on the NRC Research Press Web site at canjchem.nrc.ca on 2 June 2009. S.D. Barry, G.A. Rickard, M.J. Pushie, and A. Rauk.1 Department of Chemistry, University of Calgary, Calgary, AB T2N 1N4, Canada. 1Corresponding author (e-mail: rauk@ucalgary.ca). Can. J. Chem. 87: 942–953 (2009) shown to be a contributor to the neurotoxicity of the beta amyloid peptide of AD.6–9 The prion protein, PrP, a 231 amino acid protein, is a normal component of body tissues and is found at high concentrations in the central nervous systems of humans, as well as various other species, including mammals, avians, and yeast. Prion diseases occur when the cellular form of the protein (PrPC) is converted into its scrapie form (PrPSc), which readily aggregates. PrP selectively binds Cu(II) over other bioavailable metal ions.10 The first Cu(II)-binding region of PrP is termed the octarepeat region, which has four or more tandem repeats of the same 8 amino acids, PHGGGWGQ, beginning at Pro60 (human numbering) and repeating at Pro68, Pro76, and Pro84 in the N-terminal domain. The link between this region and the pathology of Creutzfeldt–Jakob disease (CJD) is apparent, since individuals with extra octarepeats are more prone to developing CJD.11,12 The association constants of Cu(II) binding to the octarepeat region are varied, but are generally accepted to be between 109 M–1 13 and 106 M–1.14 As in the case of the prion protein, and the beta amyloid peptide of AD, virtually all physiological copper is bound to doi:10.1139/V09-034 Published by NRC Research Press Barry et al. one or more His residues in tetracoordinated square-planar complexes. Experimentally, tetragonal coordination is preferred to pentacoordination.15 Comparison of the affinity of Cu(II) for biologically available ligands, modeled theoretically with small compounds, has shown that the imidazole group of His is a better ligand than any other,16,17 with the exception of a thiolate group (i.e., cysteine).16 However, it is well known that Cu(II) has a considerable acidifying effect on the amide N–H bond with the consequence that one or more deprotonated amide groups may be ligands to the Cu(II) ion even at physiological pH 7.18–21 Detailed information about the structures of some Cu(II)– peptide complexes is available from crystallography, but information about structures in solution comes from electron paramagnetic resonance (EPR), circular dichroism (CD), and infrared (IR) and UV–vis spectroscopies, and is much less detailed. Millhauser and co-workers were able to partially clarify the binding of Cu(II) to PrP from their use of X-ray crystallography, circular dichroism, and electron paramagnetic resonance spectroscopy studies.22–24 They determined the prominent binding modes in both solid and solvated forms at various pH values. At pH 7.4, two binding modes existed, called component 1 and component 2. The major component, component 1, has a square-planar binding environment about the Cu center consisting of three N ligands and an oxygen atom (i.e., N3O1). It was established that one of the N atoms is Np of His and the other two are deprotonated amides of the adjacent two Gly residues. The oxygen is the carbonyl oxygen of the second Gly. In the absence of the Trp residue, i.e., HGGG or HGG, component 2 is dominant. Its binding stoichiometry was tentatively identified as N2O2. These results supported previous experimentally determined binding environments at varied pH values.25,26 It was found that the minimum peptide sequence required to model the observed N3O1 and N2O2 environments of the full length Cu(II)-bound octarepeat region is the fragment ‘‘HGGG’’.22,24 Pushie and Rauk originally analyzed the binding of Cu to PrP at a high theoretical level,27 using a truncated model of the ‘‘HGGG’’ fragment in which the N- and C-terminal groups were replaced by hydrogen atoms. Nevertheless, the analysis provided some interesting insights into the interaction between Cu and PrP, including confirming the stability of the structure and binding modes determined by Millhauser and co-workers.22–24 This included identifying both component 1 and component 2, which were the most stable neutral and cationic structures, respectively. Several recent theoretical 28–30 studies of models of the octarepeat region of the prion protein have appeared since this paper was in preparation. Marino et al.28 carried out gaseous-phase calculations of the pentapeptide, Cu–HGGGW with and without an additional water molecule, on structures adapted from the crystal structure of Millhauser and coworkers (tetragonal Cu with N3O1 coordination).23 They found that the water would not occupy a fifth site in the coordination sphere of the metal but additional stabilization ensued if the Trp residue adopted a position over the square plane. Furlan et al.29 also adopted the crystal structure as a starting geometry and carried out 1 ps ab initio molecular dynamics simulations of Cu–HGGG and Cu– HGGGW surrounded by water. They found that a water 943 molecule would not occupy the fifth coordination site even in the presence of the Trp residue. Riihimäki et al. carried out MD simulations with an empirical forcefield on copper bound to the full octarepeat unit, PHGGGWGQ, also adopting the crystal structure for the tetragonal copper binding environment. With an empirical forcefield, additional coordination to copper could only arise due to electrostatic attraction. A carbonyl group was able to occupy the fifth coordination site. In the truncated HGGG model, and in the PrP octarepeats, the peptide wraps around the Cu(II) in the forward direction. This was attributed in part to the presence of the Pro residue which precludes full binding in the reverse direction. Mammalian PrP also has two additional His residues capable of Cu(II) binding within the N-terminal domain, located between the octarepeat region and the structured C-terminal domain — these are His96 and His111. The most recent reevaluation of Cu(II) coordination with this region of PrP by Jones et al. supports binding of Cu(II) at His96 and His111 in a noncooperative manner.31 At physiological pH, the Cu(II) is bound within an N3O1 or N4 coordination environment. Binding at His96 or His111 proceeds in the reverse direction — from the anchoring His residue toward the Nterminus. The objectives of this work are threefold: (1) determine the structures of the most stable species in which Cu(II) is bound to a peptide fragment containing an anchoring His residue, as a function of pH; (2) estimate the binding affinities of the structures; and (3) establish the propensity of the Cu(II) to coordinate in a forward or reverse direction relative to the His residue in the absence of specific interactions with side chains. We adopt as models, the four tetrapeptides, AcHGGGNH2 (a), AcGHGGNH2 (b), AcGGHGNH2 (c), and AcGGGHNH2 (d), to which we refer simply as HGGG (a), GHGG (b), GGHG (c), and GGGH (d), respectively. These are shown in Fig. 1a. The overlapping nature of the models affords comparison of four distinct binding modes as illustrated in Fig. 1b. In each case, the Cu(II) can bind to the His residue and may coordinate an oxygen atom or an N atom from one or more of the flanking amide groups. The amide may be protonated or deprotonated. The binding may be in the forward (toward the C terminus) or reverse (toward the N terminus) direction, and may potentially bracket the His residue (middle-forward or middle-reverse). In Fig. 1 and the following figures, all structures are shown with the Nterminal acetyl group to the left. Methods Calculations were performed using the Gaussian 03 suite of electronic structure codes.32 With several exceptions, all optimizations were performed in vacuo without using geometry or symmetry constraints, using the B3LYP hybrid functional method33 and the 6–31G(d) basis set. Harmonic frequency analysis was performed at the B3LYP/6–31G(d) level, to ensure that a minimum energy stationary point was located on the potential energy surface (no imaginary frequencies). The harmonic frequency data was used to obtain enthalpies, entropies, and zero-point energies for each structure. The zero-point energies were scaled by a factor of Published by NRC Research Press 944 Can. J. Chem. Vol. 87, 2009 Fig. 1. (a) The most stable structures of the four peptide models in water. (b) An extended polyGly peptide chain with a central His residue, residues spanned by the four modes of Cu-binding, and the relationship to the four peptide models. 0.9806. Single point energies were calculated from optimized geometries at the B3LYP/6–311+G(2df,2p) level. These energies were used to provide more accurate relative enthalpies at 0 K. The gaseous- and solution-phase entropies were calculated including the factor, Rln(n),34 where R is the normal gas constant and n is an estimate of the number of conformational degrees of freedom, based on allowing 6 conformers for a free His residue, and 5 conformers for each uncoordinated Gly residue. Values of n range from n = 1 for some tricyclic, strongly H-bonded structures, to a maximum of n = 1500 for a copper-free peptide. It is important to include an estimate of n to take into account loss of conformational degrees of freedom in the formation of cyclic structures. It should be noted that an error of a factor of 2 in the estimate of n corresponds to an error of less than 2 kJ mol–1 in the free-energy change at 298 K, but neglect of it altogether may incur an error of as much as 18 kJ mol–1 (= RTln(1500) at T = 298 K). We count only cyclic structures formed by coordination to the copper. Intramolecular hydrogen bonds are considered not to be stable in water. Values of n are given in the Supplementary data. Aqueous solvation effects were modeled by using the B3LYP/6–31G(d) wave function and the self-consistent integral equation formalism of the polarizable continuum model,35,36 as implemented in Gaussian 03 software (SCRF=IEFPCM), on the gaseous-phase-optimized geometries. The default scale factor 1.2 was used for the ‘‘united atom Hartree–Fock’’ (UAHF) radii of all neutral species. Selective scaling of UAHF radii was applied to charged species as follows: in cationic species, the radii of the Cu(II) ion and all atoms directly attached to it were scaled by 1.1; in anionic species, the radii of the Cu(II) ion and all formally neutral atoms directly attached to it were scaled by 1.1; the radii of atoms bearing a formal negative charge were unscaled (scale factor = 1.0). In the case of a deprotonated amide group, both the N and O atoms were unscaled. It is our experience that the selective scaling as described yields reasonably accurate absolute free energies of solvation of small charged species and the calculation of pKa values usually to within two units. Time-dependent (TD)37 B3LYP with the 6–31G(d) basis, in the presence of the solvent reaction field as described above, were carried out to estimate the circular dichroism (CD)38 spectrum for some of the species studied. Experimental values were adopted for the free energy of solvation of the proton and water: DGsolv(H+) = –1107 kJ mol–1 39; DGsolv(H2O) = –16.4 kJ mol–1. The value for water corresponds to the transfer of a water molecule from the gaseous phase at a concentration of 1 mol/L into aqueous medium at 55 mol/L.40 pKa values reported in the text and figures were calculated from the aqueous free-energy change of the acid ionization reaction, AHðaqÞ mþ ! AðaqÞ ðm1Þþ þ HðaqÞ þ . We expect that for the large systems, at the level of theoretical treatment described above, the residual errors in aqueous free-energy changes are likely to be approximately ±15 kJ mol–1. This range corresponds to about ±2 pKa units. Thus we will place emphasis only on differences that are larger than these amounts. Published by NRC Research Press Barry et al. 945 Fig. 2. GHGG(b)/Cu Structures. Ball colours: green = Cu2+; orange = C; blue = N; red = O. Free-energy changes, DG, are in units of kJ mol–1 in aqueous solution. Table S1 of Supplementary data contains all primary calculated data for all species discussed below. Table S2 contains calculated CD data. The Cartesian coordinates of all species are also provided in the Supplementary data. Results and discussion Many conformations of each system were examined to find the most stable in solution. In most cases, the most stable conformation in the gaseous phase involved collapsed structures with a maximum number of intramolecular hydrogen bonds. However, these were often not the most stable structures in solution after addition of DGsolv; the solvent reaction field strongly favoured extended structures with a maximum exposure of the amide groups to the solvent. In the case of 1b and 2b (Fig. 2), there were no local minima corresponding to extended conformations in the gaseous phase, but in each case, geometry optimization with the solvent reaction field yielded a more stable conformation in solution. For these, the zero-point energy and thermal corrections were taken from the nearest gaseous-phase minimum. The binding of Cu(II) to the peptides is strongly dependent on pH. The pKa of a protonated His residue is approximately 7 for loss of proton from either side chain N atom. At pH < 7, both N atoms of the imidazole group are protonated, and the Cu(II) ion must compete with a proton for binding to one of the N atoms. At the present level of theory, the complexes of copper ion with water and other simple ligands are best described as tetracoordinated, having a distorted square-planar coordination sphere.16 Since an amide carbonyl oxygen will probably not displace water from the coordination sphere, initial binding of the copper will be to one of the imidazole N atoms provided the freeenergy penalty for deprotonating the N atom (= –RT(ln(Ka) – ln[H+]) is not greater than the free energy of binding. The structures of the most stable complexes of Cu(II) with each peptide, according to the reaction, ½1 Cu2þ ðH2 OÞ4ðaqÞ þ LðaqÞ ! Cu2þ ðH2 OÞ3 LðaqÞ þ H2 OðaqÞ are designated 1a, 1b, 1c, and 1d, and are shown in Figs. 3 or 4, 2, 5, and 6, respectively. In each case, the coordination is through Nt yielding a tetracoordinated metal center. It is of interest to note that structures that are substantially more stable than 1a, 1b, 1c, and 1d in the gaseous phase were found by attaching the copper ion to Np. In these cases (not shown), the copper ion is pentacoordinated. Although not part of the starting structure, the His carbonyl O atom moved into the fifth coordination site upon geometry optimization, and short H-bonds were formed between two coordinated water molecules and other carbonyl O atoms. However, the Np-coordinated structures had significantly lower free energy of solvation, with the result that, for all systems, the most stable structures in water, where a single water is displaced, involved coordination of the aqueous copper ion through Nt. with the peptide backbone in an extended configuration. Since the structures are isomeric, it is possible to compare their energies. The relative DG(aq) of 1a, 1b, 1c, and 1d, are Published by NRC Research Press 946 Can. J. Chem. Vol. 87, 2009 Fig. 3. HGGG(a)–Cu structures. Ball colours: green = Cu2+; orange = C; blue = N; red = O. Free-energy changes, DG, are in units of kJ mol–1 in aqueous solution. 0, 5, 23, and 11 kJ mol–1, respectively. We return to a discussion of the low-pH attachment of Cu(II) below when we compare the results with experiment. The remainder of the structures discussed here are those expected to exist at higher pH. In all of these, the Cu(II) is bound to the histidine via the Np atom of the imidazole group. The Np atom is close enough to the peptide backbone to allow easy access to the Cu centre by amide groups that lie adjacent to the Ca of the histidine residue and in forward and reverse directions down the chain. The sequence of models permits evaluation of the energetics of chelation of the Cu in both the forward direction (toward the C terminus) and the backward direction (toward the N terminus) as illustrated in Fig. 1. We consider structures in which the Cu– peptide complex has lost a single proton (structures 2x), two protons (structures 3x), and three protons, either from three amide groups (4x), or from two amide groups and Nt of the imidazole (5x). Here x is one of a (HGGG series), b (GHGG series), c (GGHG series), or d (GGGH series). For convenience, all of the structures in all of the figures are oriented with the N-terminus to the left and the C- terminus to the right. We briefly discuss each of the tetrapeptides separately, and then compare them. HGGG structures The sequence of the most stable structures of each type in the HGGG series resulting from successive proton loss, namely 1a, 2a, 3a, 4a, and 5a, is shown in Fig. 3. Several conformations of the copper-free HGGG peptide, HGGG(a), were found. The most stable of these in solution, the Npprotonated tautomer, is shown in Fig. 4. It attaches to CuðaqÞ 2þ with low affinity, DG(2) = –6 ± 12 kJ /mol–1 to yield structure 1a in which the Cu(II) is tetracoordinated and bound to Nt of the imidazole. ½2 CuðH2 OÞ4 2þ þ HGGGðaÞ ! 1a=CuðH2 OÞ3 2þ þ H2 O Successive loss of water leads to the other structures shown in Fig. 4. The most stable structure in which two waters of coordination remain, 1a’, is unstable relative to the separated species (left hand side of eq. [2]) by 33 kJ mol–1. Successive loss of the remaining two waters of coordination yields 1a@ and 1a’@, that are bound relative to the separated species by DG(aq) = –6 kJ mol–1 and –17 kJ mol–1, respectively. In each of 1a’, 1a@, and 1a’@, the Cu(II) is pentacoordinated, with a tetragonal pyramidal geometry. Structure 2a (Fig. 3) is the most stable singly-deprotonated species. Of the many structures examined here, 2a is unusual in that it has a ‘‘middle-forward’’ bonding configuration (see Fig. 1b). The pentacoordination in 2a is probably weak. The average distance for a Cu–O bond at this level of theory is 2.00 ± 0.02 Å. The length of the fifth bond (to the O=C of His) is 2.49 Å, indicating that it is considerably weaker and may not survive in aqueous solution. Structure 2a has nominally an N2O2 coordination pattern. It can be identified with structure II of Bonomo et al.,21 and ‘‘component 2’’ of Published by NRC Research Press Barry et al. 947 Fig. 4. Attachment of aqueous Cu(II) to HGGG(a) with successive loss of water. The most stable structures of each type are shown. Ball colours: green = Cu2+; orange = C; blue = N; red = O. Free-energy changes, DG, are in units of kJ mol–1 in aqueous solution. Millhauser and co-workers, and confirms their tentative N2O2 assignment.41 The second most stable singly deprotonated structure, 2a’ (Fig. 3), also has N2O2 coordination and is predicted to be less stable by 18 kJ mol–1. As is evident from both structures 2a and 2a’, the first loss of a proton is predicted to occur from the N–H bond of the second Gly residue, since this permits the two strong electron pair donors, the amide group and the imidazole group, to be trans to each other in the Cu coordination shell. Structure 2a is predicted to have pKa = 8 ± 2, corresponding to ionization of the N–H bond of the first Gly residue, yielding 3a. Species 3a has N3O1 coordination in the ‘‘forward’’ direction, with two five-membered rings involving the two deprotonated amide groups and a coordinating carbonyl O atom, and a seven-membered ring involving the His residue. A second structure, 3a’ (Fig. 3) with the same coordination pattern as 3a, but with the seven-membered ring inverted, is predicted to be slightly less stable. In 3a’, the oxygen atom of the N-acetyl group almost makes a fifth ligand to the copper ion. Either or both of 3a and 3a’ are candidates for structure III of Bonomo et al.,21 and ‘‘component 1’’ of Millhauser and co-workers.41 Structure 3a has four remaining sites for deprotonation, three from backbone N–H bonds and the Nt–H bond of the imidazole. The latter is calculated to be the most acidic, with pKa = 10 ± 2, yielding structure 5a. We could find no backbone N–H bond with pKa < 17, i.e., corresponding to structure 4a. Thus our results suggest that the third deprotonation is from the imidazole group rather than from the backbone. We return to this apparent disagreement with the experimental observations by Bonomo et al.21 in the ‘‘Relationship to experimental studies’’ section below. GHGG structures In a fashion analogous to the HGGG (a) system, the pHdependent origin of GHGG (b) structures is displayed in Fig. 2. The first attachment to the copper ion yields 1b with binding affinity, –23 kJ mol–1. Despite the availability of two amide groups preceding the His residue, the most stable coordination pattern, after loss of a single proton and three water molecules, is in the ‘‘forward’’ direction, yielding 2b with N2O2 coordination. As in the case of 2a, loss of the proton has occurred from the ‘‘i + 2’’ Gly residue, affording a trans orientation of the two N ligands. Loss of the proton from the ‘‘i + 1’’ Gly residue of 2b, yielding 3b, is predicted to occur more easily than in the case of 2a, with pKa = 6 ± 2. Structure 3b is predicted to deprotonate from Nt–H of the imidazole with pKa = 12 ± 2, yielding 5b. Of the structures that could be derived by further deprotonation of a backbone N–H bond, the most stable is 4b, which has an N3O pattern of coordination and three fivemembered rings. The coordination direction of 4b is ‘‘middle-reverse’’ and the His residue is not involved. Structure 4b is predicted to be 94 ± 10 kJ mol–1 less stable than 3b (at pH = 7, DG(aq)(3b ? 4b + H+) = 54 ± 10 kJ mol–1) and 27 kJ mol–1 less stable than 5b. GGHG structures The pH-dependent origin of GGHG(c) structures is displayed in Fig. 5. As was the case with 2a, the most stable coordination pattern, after loss of a single proton and three water molecules from 1c, is a pentacoordinated ‘‘middle’’ structure, 2c. However, 2c differs in detail from 2a. In 2c, the deprotonated amide N–H bond is that of the preceding (i.e., i – 1) Gly residue, again leaving the two strong N donors Published by NRC Research Press 948 Can. J. Chem. Vol. 87, 2009 Fig. 5. GGHG(c)–Cu structures. Ball colours: green = Cu2+; orange = C; blue = N; red = O. Free-energy changes, DG, are in units of kJ mol–1 in aqueous solution. in a trans orientation in the copper coordination shell. Cis to the imidazole group is a neutral N atom of the His residue. The two remaining ligands are O atoms of the i – 2 Gly residue and of the His residue. The Cu–O bond lengths are 1.99 Å and 2.30 Å, respectively, indicating that the latter ligand is less strongly held and would probably be released in solution. Within 2 kJ mol–1 of 2c is a second pentacoordinated structure, 2c’, that differs in that the protonation states of the His and i – 1 Gly residues are reversed and the fifth coordinating O ligand is from the amidated C-terminus (formally the O atom of the i + 2 residue). Loss of the proton from the N atom of the His residue of 2c (or the i – 1 Gly residue of 2c’) yields the neutral structure, 3c, which also has the strong amide donor opposite the imidazole group and four-coordination with N3O1 ligand distribution in the reverse direction. Loss of the proton from 2c or 2c’, yielding 3c, is predicted to occur very easily, with pKa = 5 ± 2. Structure 3c is predicted to deprotonate primarily from the i – 2 Gly residue (pKa = 10 ± 2), yielding 4c, and secondarily from Nt–H of the imidazole (pKa = 12 ± 2), yielding 5c. Structure 4c has an N4 pattern of coordination with two five-membered rings and a six-membered ring. GGGH structures The pH-dependent origin of GGGH(d) structures is displayed in Fig. 6. The singly deprotonated structure 2d is entirely analogous to 2c’, except that it is four-coordinated in the reverse direction, rather than five-coordinated in the ‘‘middle-reverse’’ direction. In this case, the shorter C-terminus does not provide a weak fifth-coordinating ligand. Deprotonation of 2d occurs with pKa = 5 ± 2 to yield 3d, which has the same bonding characteristics as 3c, namely coordination in the reverse direction with ligand pattern N3O1. Further deprotonation of 3d is also entirely analogous to 3c, with almost equal probability for proton loss from Nt–H and i – 2 Gly residue, with the similar pKa values, pKa = 12 ± 2, and pKa = 13 ± 2,yielding 4d and 5d, respectively. Like 4c, structure 4d has an N4 pattern of coordination with two five-membered rings and a six-membered ring. Structural comparisons Isomeric structures may be compared to each other. The relative aqueous free energies DG(aq) of the most stable singly deprotonated species described above are (in kJ mol–1): 2a, 1; 2b, 0; 2c, 34; and 2d, 33. The first two represent binding in the forward direction with configuration N2O2. The last two structures are bound in the reverse direction. Thus, in the case of singly deprotonated structures, binding in the forward direction is preferred. The relative aqueous free energies DG(aq) of the most stable doubly deprotonated species described above are (in kJ mol–1): 3a, 15; 3b, 0; 3c, 29; and 3d, 27. Again, the first two represent binding in the forward direction, but with configuration N3O1. The last two structures are bound in the reverse direction, also with configuration N3O1. Thus, the neutral doubly deprotonated structures also prefer to bind in Published by NRC Research Press Barry et al. 949 Fig. 6. GGGH(d)–Cu structures. Ball colours: green = Cu2+; orange = C; blue = N; red = O. Free-energy changes, DG, are in units of kJ mol–1 in aqueous solution. the forward direction. The difference is most pronounced in the cases of GHGG(b) and GGHG(c), which could bind in either direction. The more stable structure 3b has a five, five, seven ring system while the less stable structure 3c has a six, five, seven ring system. The calculated difference in the enthalpies of the two ring systems is DH298 = 14 kJ mol–1. The remaining 15 kJ mol–1 of the difference originates primarily from the higher entropy (–TDS = 10 kJ mol–1) and slightly more favourable solvation (DDGsolv = 5 kJ mol–1) of 3b, both contributions of which are due to the longer free peptide chain in the reverse direction. The relative aqueous free energies DG(aq) of the most stable anionic triply backbone-deprotonated species described above are (in kJ mol–1): 4a, 25; 4b, 5; 4c, 0; and 4d, 5. There is no significant difference in stability among the last three structures. Structures 4c and 4d exemplify reverse binding and have an N4 coordination pattern. Both structures are favoured by enthalpy over structure 4a, DH298 = –18 kJ mol–1 and DH298 = –24 kJ mol–1, respectively. Structure 4b is technically a middle-reverse binding pattern that has lost the imidazole as a ligand. It is the least stable by enthalpy in the gaseous phase, but a significantly higher free energy of solvation brings it into the same range as the reverse binding structures. Thus, the calculations suggest that at high pH there is a clear preference for backbone deprotonation in the reverse direction, which yields a five, five, six ring pattern as opposed to the five, five, seven ring pattern of forward binding.18 Relationship to experimental studies Protonated imidazoles of His residues have pKa values in the range 6–7.42 The calculated pKas of the present systems are: HGGG(a)H+, 7.5; GHGG(b)H+, 12; GGHG(c)H+, 6;. GGGH(d)H+, 5. Thus, the values of three of the His residues lie within the error in pKa values (±2) expected due to uncertainties in relative aqueous free energies, but the His residue of GHGG(b)H+ is outside of these bounds. The reason for this is unclear. The capping at both ends of the peptide models permits direct comparison with previous spectroscopic and potentiometric titration studies on AcHGGGNH2.21 Similar studies have also been carried out on the related systems, AcHGGG, AcGHGG, AcGGHG, and AcGGGH, in which the Cterminus was a free carboxylate group.20 The calculated free energies of binding (eq. [1], DG(1) are (kJ mol–1): 1a, –6; 1b, –23; 1c, –8; 1d, –17. The binding free energy of the first and third is close to zero; 1b and 1d are predicted to be bound in water. An experimental value, –23 kJ mol–1, is available21 for the first coordination of Cu(II) to HGGG (a) yielding a 1:1 complex with +2 charge. The number of waters of coordination is unknown from the experiment. In the case of the HGGG(a) system, we examined the successive loss of water for the Cu(II)-bound system at low pH (i.e., with net charge = +2). The results are displayed in Fig. 4. The binding affinity of the most stable structure, 1a’@, DG = –17 ± 10 kJ mol–1, is in satisfactory agreement with exPublished by NRC Research Press 950 periment, but is also within the overlapping error bars of 1a. We conclude that it is not possible to determine computationally the degree of hydration of the species arising from the first attachment of the Cu(II) ion to HGGG(a) with the present level of theory. Consequently, we have not pursued this course with any of the other three systems, but rather take the Nt-bound Cu(II)(H2O)3 species 1b, 1c, and 1d, as being representative. For two of these, the calculated binding affinity 1b, DG = –23 kJ mol–1and 1d, DG = –17 kJ mol–1, agree well with the experimental values based on the stability constant reported for the 1:1 complex of Cu(II) with AcGHGG and AcGGGH.20 In the pH range 6.5–8.5, two Cu–AcHGGGNH2 complexes, corresponding to singly and doubly deprotonated species, were observed potentiometrically and spectroscopically.21 The relative abundance of the former was always less than 15% . These may be identified with 2a and 3a, respectively (Fig. 3). The pKa for loss of the second proton, 6.25, is in satisfactory agreement with that calculated for 2a ? 3a + H+, 8 ± 2. ESI-MS revealed a signal corresponding to protonated and sodiated forms of a neutral 1:1 Cu– AcHGGGNH2 complex that notably did not contain any coordinated water molecules. A recent ESI-MS study by one of us confirmed this observation.43 Two structures consistent with EPR and MS data were proposed for the neutral species, both of N3O1 square-planar coordination in the tetragonal plane, but differing by which terminal carbonyl oxygen atom formed the fifth coordination site.21 The present results suggest that the neutral complex corresponds to 3a, which has a square-planar N3O1 coordination geometry, but does not have involvement of either terminal carbonyl oxygen in the coordination sphere of the copper. In solution, this site would be transiently occupied by a water molecule. Similar potentiometric measurements were carried out on the free terminal carboxylate system, Cu–AcHGGG, by Orfei et al.,20 with very similar results. In that case, pKa = 6.96 was assigned for the loss of the second proton. These authors carried out gaseous-phase modeling using the semiempirical PM3 method on the ‘‘neutral’’ species (actually negatively charged because of the carboxylate group) and obtained the same square-planar N3O1 coordination geometry as in 3a, with coordinating water ligands above and below the plane.20 In our high-level ab initio study, the fifth and sixth ligands to Cu are not stable to exchange in solution. Both Bonomo et al.21 and Orfei et al.20 detected a third ionization with pKa = 8.9, yielding a species with a negative charge (ignoring the charge of the carboxylate group). Both groups interpreted the EPR spectra as consistent with N4 coordination, in which three deprotonated amide nitrogen atoms, as well as imidazole N atom were coordinated to copper in a square-planar arrangement. ESI-MS signals collected in negative mode proved the absence of water.21 As shown in Fig. 3, we could find no species with N4 coordination in an accessible pH range. The lowest pKa for loss of a third amide proton from 3a is calculated to be pKa = 17, yielding 4a. The lowest energy negatively charged species originates from loss of the remaining proton of the imidazole group, namely 5a, which is formed from 3a with pKa = 10 ± 2. The calculated pKa is essentially equal to the observed pKa. Thus our data suggest that it is the imidazole that is the source of the third deprotonation, rather than a Can. J. Chem. Vol. 87, 2009 third amide group. As we show below, this conclusion is also supported by CD data. We have no explanation for the discrepancy with the EPR results. As well as AcHGGG, Orfei et al., also examined the pHdependent evolution of structures of Cu(II) complexes with AcGHGG, AcGGHG, and AcGGGH.20 The stability constants for formation of a putative 1:1 Cu–peptide complex without loss of any protons were similar among all of the species, and correspond to free-energy changes in the narrow range –25 kJ mol–1 to –27 kJ mol–1, as mentioned above. For each of these, the loss of the first proton produced a species in minor abundance compared to that derived from loss of two protons. pKa values for both processes for all systems were in the narrow range of 6–7. We would assign structures 2b, 2c, and 2d to the former and 3b, 3c, and 3d, to the latter. However, we find some variability in the energetic parameters among the three systems, and the calculated data for the Cu(II)–GHGG(b) system, shown in Fig. 2, does not fit the observed pattern for the free terminal carboxylate systems. Experimentally, the third ionization in the case of the Cu–AcGHGG system was anomalous but the first two were not. In the case of Cu– AcGGHG and Cu–AcGGGH, as for Cu–AcHGGG, the third ionization occurred with a pKa below 9 units, but the third ionization in the AcGHGG system had an apparent pKa 2 units higher, and was attributed either to ionization of a bound water molecule or the imidazole.20 An N4-coordinated pattern was not seen by EPR. In our study, the Cu– GHGG(b) system is unusual in several respects. Single and double deprotonation, yielding 2b and 3b, respectively, is predicted to occur at pH < 6. The most acidic proton of 3b is found to be the imidazole proton, as it was in the case of 3a but 2 pKa units higher. The lowest energy structure that has three amide groups deprotonated is 4b, in which the imidazole group is not coordinated to the Cu(II). This complex is 27 kJ mol–1 higher in energy than 5b. By way of contrast, in both the Cu–GGHG(c) (Fig. 5) and Cu–GGGH(d) (Fig. 6) systems, the third ionization occurs with slightly higher probability from a third amide than from the imidazole group, and with very similar acidity constants. Circular dichroism data Orfei et al.20 found that the high-pH CD spectra of the Cu–AcGGHG and Cu–AcGGGH systems displayed evidence of an identical chromophore. The spectra are dominated by a pair of oppositely signed bands of nearly equal intensity: Cu–AcGGHG, 600 nm (–), 500 nm (+); Cu– AcGGGH, 600 nm (–), 500 nm (+). Distinctly different chromophores were observed in the CD spectra at high pH of Cu–AcHGGG and Cu–AcGHGG. These both showed negative rotatory strength above 700 nm, a positive band near 600 nm, and a negative band near 520 nm. To seek an explanation for the different CD spectra at high pH, we applied TD-B3LYP/6–31G(d) methodology to calculate and simulate the aqueous CD spectra of the triply deprotonated amide structures, 4a, 4c, and 4d, as well as the imidazole deprotonated forms, 5a, 5b, 5c, and 5d, and the imidazole-released form, 4b, in the spectral range studied by Orfei et al. The simulated CD spectra are displayed in Fig. 7, using Gaussian lineshapes with a half width of 0.37 eV (100 nm for a peak centred at 600 nm). Seven to Published by NRC Research Press Barry et al. Fig. 7. Simulated CD spectra by TD-B3LYP/6–31G(d) in the presence of solvent reaction field: (a) HGGG structures 4a and 5a; (b) GHGG structures 4b and 5b; (c) GGHG structures 4c and 5c; (d) GGGH structures 4d and 5d. ten electronic transitions are predicted to occur on the spectral range 300–700 nm. The simulated CD spectra of the N4 species 4c and 4d are very similar to each other, but are qualitatively different from 4a. The former have negative rotatory strength in the range 450–700 nm, while 4a has a bisignate feature in the range with positive rotatory strength 951 near 500 nm. 4b is different again, with a negative CD band at 490 nm and positive intensity above 520 nm. The imidazole-deprotonated systems are distinct from the N4 systems. The spectra of 5c and 5d are almost identical to each other with CD pattern (right to left) weak (+), weak (–), medium (+), strong (–), and medium (+). The parallel systems 5a and 5d also have a similar pattern but have larger CD intensity in the 450–700 nm range. Because of the limitations of the theoretical method and the fact that the systems are different, we do not expect a perfect match between our simulated spectra and the experimental spectra of Orfei et al. Nevertheless, some of the computed spectra reproduce the main spectral features of the experimental ones very well. For the N4 systems, 4c, and 4d (dashed lines in Figs. 7c and 7d), the dominant characteristic is bisignate, as are the experimental high-pH CD spectra of Cu–AcGGHG and Cu–AcGGGH, with positive rotatory strength to short wavelength and negative to longer. The calculated spectra are more complex in appearance than the experimental spectra, but reveal the multiple-transition origin of the deceptively simple spectra. The calculated spectra for the imidazole-deprotonated species 5c and 5d do not match the experimental spectra, thereby confirming the N4 coordination pattern for the high-pH species of the GGHG and GGGH species. In the case of the GHGG system, the simulated CD spectrum of 5b exactly matches the sign sequence of the experimental high-pH spectrum of Cu–AcGHGG although all the peaks are shifted to shorter wavelength. The spectrum of 4b is very different. Thus the spectroscopic as well as energetic calculations are consistent with an imidazole-deprotonated structure for this system. The most troublesome system is HGGG at high pH. Both Bonomo et al.21 and Orfei et al.20 found clear indications of N4 coordination in Cu–AcHGGGNH2 and Cu–AcHGGG, respectively, by EPR. However, our calculations suggest that the most stable N4 system, 4a, is energetically disfavoured compared to the imidazole-deprotonated species, 5a, which has N3O1 coordination. Also the CD data of Orfei et al.20 are quantitatively different from Cu–AcGGHG and Cu– AcGGGH which were clearly assigned as N4 above. The simulated CD spectrum of 4a is also quantitatively different from that of 4c and 4d. The difference is probably due to the different direction of coordination, forward, as apposed to reverse. In our opinion, there is a better match between the experimental CD spectrum of Cu–AcHGGG obtained by Orfei et al and our simulated spectrum of 5a. Thus, our energetic and spectroscopic calculations both point to an imidazole-deprotonated structure, 5a, for both Cu–AcHGGG and Cu– AcHGGGNH2 at pH > 11. The discrepancy between the EPR and CD data could be resolved if the different spectroscopic techniques were sensitive to different species in solution. However, we have been unable to find an N4 species analogous to 4a in an accessible energy range. Summary and conclusions The structures and relative free energies in aqueous solution of the Cu(II) complexes of the peptides, AcHGGGNH2 (HGGG(a)), AcGHGGNH2 (GHGG(b)), AcGGHGNH2 (GGHG(c)), and AcGGGHNH2 (GGGH(d)) were deterPublished by NRC Research Press 952 mined as a function of pH. Numerous structures of each species were found by gaseous phase geometry optimization and the effect of solvation estimated by the IEFPCM continuum solvation model. Free energies of solvation of the ionic species are large and favour structures with an extended peptide chain. It is rare that the most stable gaseous-phase structure is also the most stable in the aqueous phase. The solvation procedure gives satisfactory results for neutral and cationic species, but undersolvates anionic species, as judged against experimental observations at pH ‡ 8. At low pH, the tetraaquo Cu(II) ion attaches to a single N atom of the imidazole group with the expulsion of one or more waters of coordination. In Cu–HGGG(a), for which all possible levels of hydration were examined, the most stable structure, 1a@’, had no waters in the copper coordination shell, a result consistent with ESI-MS.21,43 In all Cu(II)–peptide complexes, deprotonation of two amide groups occurs readily at or even below physiological pH = 7. The first N–H bond to ionize is that which places the amide N atom in a trans orientation to the imidazole N atom, in the forward direction in the case of HGGG(a) and GHGG(b) (the i + 2 Gly), and in the reverse direction in the case of GGHG(c) and GGGH(d) (the i – 1 Gly). The intervening amide group in protonated form and the carbonyl of the i ± 2 Gly complete the coordination sphere of the Cu(II). The second ionization then occurs from the intervening amide group to yield a neutral 1:1 complex with N3O1 coordination pattern as the most abundant species at pH = 7. In the case of both singly and doubly deprotonated structures, binding in the forward direction is preferred. The observation of forward binding in the crystal structure of the octarepeat region of the prion protein was tentatively attributed to the blocking effect of the proline residue immediately preceding the His. The present results suggest that forward binding would be the preferred mode in any case. It should be noted however, that it is not possible to determine the effect of side-chain interactions on the direction of binding from the present models. The computed relative free energies of the various ionized species in the Cu–HGGG(a) system are in good agreement with the experimental potentiometric studies of Bonomo et al.,21 with the exception that ionization of the third proton, which was attributed to a third amide group and observed at pH & 9, is calculated to come from the imidazole at similar pH (pKa = 10). Simulated CD spectra of the candidate highpH species support this result. Supplementary data Supplementary data for this article (Table S1: Primary computed data for all species: energies, zero-point energies, thermal corrections to the enthalpy, entropies, numbers of conformations and free energies of solvation; Table S2: CD spectral data for 4a, 5a, 4b, 5b, 4c, 5c, 4d, and 5d) are available on the journal Web site (canjchem.nrc.ca) or may be purchased from the Depository of Unpublished Data, Document Delivery, CISTI, National Research Council Canada, Ottawa, ON K1A 0R6, Canada. DUD 3933. For more information on obtaining material, refer to cisti-icist. nrc-cnrc.gc.ca/cms/unpub_e.shtml. Geometries are available upon request. Can. J. Chem. Vol. 87, 2009 References (1) This article is part of a Special Issue dedicated to Professor T. Ziegler. (2) Dobson, C. M. Trends Biochem. Sci. 1999, 24 (9), 329–332. doi:10.1016/S0968-0004(99)01445-0. PMID:10470028. (3) Gaggelli, E.; Kozlowski, H.; Valensin, D.; Valensin, G. Chem. 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