Understanding Regioselective Cleavage in Peptide Hydrolysis by a

J. Phys. Chem. B 2010, 114, 8525–8535

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Understanding Regioselective Cleavage in Peptide Hydrolysis by a Palladium(II) Aqua Complex: A Theoretical Point of View Violeta Yeguas,† Pablo Campomanes,‡ Ramoń Lo´pez,*,† Natalia Dıáz,† and Dimas Sua´rez† Departamento de Quı´mica Fı´sica y Analı´tica, UniVersidad de OViedo, C/Juliań ClaVerıá, 8, 33006 OViedo, Spain, and Laboratory of Computational Chemistry and Biochemistry, Ećole Polytechnique Fe´de´rale de Lausanne (EPFL), CH-1015 Lausanne, Switzerland ReceiVed: March 2, 2010

Hydrolytic cleavage of the oligopeptides Ace-Ala-Lys-Tyr-Gly∼Gly-Met-Ala-Ala-Arg-Ala and Ace-LysGly-Gly-Ala-Gly∼Pro-Met-Ala-Ala-Arg-Gly by [Pd(H2O)4]2+ was theoretically investigated by using molecular dynamics simulations and quantum mechanical calculations. The Pd anchorage to the peptide sequence is crucial to provoke the cleavage of the second bond upstream from the anchored methionine. For both cases, the most favorable reaction mechanism is a three-step route. The first step coincides with the experimental suggestion found for the Gly∼Pro-Met sequence on a cleavage caused by an external attack of a water molecule to a complex in trans conformation of the scissile Gly∼Gly and Gly∼Pro peptide bonds. However, our results uncover the important role played by the presence of a Pd-coordinated water molecule, which simultaneously interacts with the carbonyl oxygen atom of the Gly amino acid in the Gly∼Gly and Gly∼Pro bonds. In accordance with experimental facts, the rise of the hydrolysis reaction rate when the Pro amino acid is located in the scissile peptide bond was also corroborated. The findings obtained at a molecular level from the present computations not only are relevant to rationalize the previously reported experiments but also could be of importance in designing new Pd(II) complexes for the regioselective cleavage of peptides and proteins. Introduction Selective cleavage of peptides and proteins is a very important issue due to its implication in many bioanalytical and bioengineering applications such as protein sequencing, peptide mapping, protein footprinting and folding studies, protein semisynthesis, and purification of fusion proteins.1-12 Hydrolysis of the amide linkage in peptides and proteins is the most preferred cleavage method because the reaction products, amines and carboxylic acids, can be condensed into new products or otherwise can be chemically modified. However, it is wellknown that this kind of hydrolytic process requires relatively long reaction times. For instance, the half-life for hydrolysis of the amide linkage in Gly∼Gly is 350 years at neutral pH and 25 °C.13 Therefore, different strategies have been undertaken to efficiently hydrolyze peptide bonds.14-17 One of them implies the use of proteolytic enzymes,1,18 such as pepsin, chymotrypsin, thermolysin, or pronase, which present an impressive catalytic power, but several shortcomings have been addressed in the literature. For example, the broad substrate specificities exhibited by many of these enzymes make them inconvenient for use in sequencing experiments. Chemical reagents like cyanogen bromide, O-iodosobenzoate, and hydroxylamine are an alternative strategy to enzymes but require harsh reaction conditions and often produce incomplete cleavage and relatively low yields.19,20 Transition-metal complexes are also used for cleaving peptides and proteins under nondenaturing conditions of temperature and pH.9,11,12,14-16,20-34 Because mild conditions can be employed, these reagents show great promise for use in * Corresponding author. E-mail: [email protected]. † Universidad de Oviedo. ‡ ´ Ecole Polytechnique Fe´de´rale de Lausanne (EPFL).

different biochemical applications. Thus, there is a growing interest in the design and synthesis of protein cleaving metal complexes. It has been reported that complexes of Ce(IV), Co(II), Co(III), Cu(II), Mo(IV), Ni(II), Pd(II), Pt(II), Zn(II), and Zr(II) cleave hydrolytically the amide linkage in peptides.9,17,27-32 Many of these experimental studies have focused on the Pd(II) and Pt(II) complexes20,35-58 because they are extremely useful reagents for sequence-specific hydrolysis of peptides and proteins.20,37,46,49,52,59,60 Among them, a recent study on the hydrolytic cleavage under mild conditions of several peptide sequences (Ace-Ala-Lys-Tyr-Gly∼GlyMet-Ala-Ala-Arg-Ala, Ace-Lys-Gly-Gly-Ala-Gly∼Pro-Met-AlaAla-Arg-Gly, Ace-Lys-Gly-Gly-Ala-Gly∼Pro-His-Ala-Ala-ArgGly, etc.) by [Pd(H2O)4]2+ has shown that the presence of the Pro amino acid accelerates the reaction rate with respect to the peptide hydrolyses in which this residue is not present.20 In accordance with previous experimental studies,46,52,60 for all the investigated processes, [Pd(H2O)4]2+ spontaneously binds to the side chains of the Met or His residues and regioselectively promotes hydrolytic cleavage of the second amide bond upstream from this anchoring residue, that is, for instance, the Gly∼Gly and Gly∼Pro bonds in the Gly∼Gly-Met and Gly∼Pro-Met (or Gly∼Pro-His) peptide sequences, respectively (see Scheme 1). These processes start with the complexation of the peptide, in which the metal ion anchors to the sulfur or nitrogen atoms of the Met or His residues, respectively. Then, the Pd(II) ion deprotonates the secondary amide group and binds to the nitrogen atom of the resulting amidate anion, thus giving the so-called hydrolytically active complex. With regards to the reaction mechanism on the hydrolysis of the peptide bond by the Pd(II) ion, two general mechanistic proposals were considered by the authors as in previous experimental studies.20,46,51 In one, the anchored Pd(II) complex internally delivers an aqua ligand to the proximate amide group. In the other mechanism, the

10.1021/jp101870j  2010 American Chemical Society Published on Web 06/09/2010

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SCHEME 1: Binding of the Pd(II) Ion to the Met Side Chain and Stepwise Coordination of the Deprotonated NH Group in the Peptide Backbone Upstream from the Anchora

a

For simplicity, the Pro-Met-peptide sequence is shown.

SCHEME 2: Possible Hydrolytically Active Complexation Modes Proposed by Experimentalists along with the Inclusion of the trans Conformation of the Binding Mode 1a

a

The Gly∼Gly-Met case is displayed as an illustration.

Pd(II) complex interacts with the carbonyl oxygen atom and thus activates the scissile amide group toward nucleophilic attack by an external water molecule. The former mechanism is frequently ascribed to the cis conformation of the scissile peptide bond in the Pd(II)-peptide complex (see cis conformation of binding mode 1 in Scheme 2), while the trans one is usually invoked in the latter case (see binding mode 2 in Scheme 2). The external attack mechanism was confirmed for the Pro-containing peptides based mainly on the detection of a trans conformation of the Gly5-Pro6 peptide bond.20 As a consequence, they deduced that the carbonyl oxygen atom of the Gly amino acid in the hydrolytically active complex should be oriented toward the Pd(II) ion. Although no evidence was found for the operative mechanism in the Gly∼GlyMet peptide sequence, the internal delivery mechanism starting from the cis conformation of the Gly-Gly bond instead of a trans one (see binding modes type 1 in Scheme 2, respectively) was suggested due to the lower basicity of the amide carbonyl oxygen atom of the scissile bond in Gly∼Gly-Met compared to that in Gly∼Pro-Met.20 Although significant progress has been made in the Pd(II) complexes, the design of new regioselective reagents that efficiently catalyze the hydrolysis of amino acid sequences in diverse sets of folded proteins could be improved through the potentially useful information provided by theoretical studies on the reaction mechanisms involved in these processes. In fact,

Yeguas et al. a density functional theory (DFT) investigation based on the optimization of conformations has permitted us to propose a reaction mechanism, which explains the nature of the selective cleavage of the His18∼Thr19 bond in cyctochrome c promoted by Pd(II) complexes.61 Another interesting work has also reported an interpretation of the stereochemical requirements for the efficient cleavage of His-containing peptides by Pd(II) complexes by finding several appropriate conformations between the dipeptide Ace-HisGly and Pd(H2O)32+ through molecular dynamics simulations.43 The mechanism of the [Pd(H2NC2H4NH2)]2+-catalyzed hydrolysis of the amide N-formyltryptophanamide as a very simple model of tryptophan-containing peptides was theoretically investigated by means of DFT calculations as well.62 These previous studies have reported some insights into the regioselective hydrolytic cleavage of peptides promoted by Pd(II) complexes. To date, however, there is not yet a theoretical mechanistic investigation involving the location of more realistic critical structures (reactant complexes, transition states, etc.). Quantum mechanical (QM) calculations in conjunction with statistical simulations would be necessary to achieve a deeper understanding of experimental facts. All of this prompted us to undertake a theoretical study on the [Pd(H2O)4]2+-catalyzed cleavage of the synthetic peptides Ace-Ala-Lys-Tyr-Gly∼GlyMet-Ala-Ala-Arg-Ala and Ace-Lys-Gly-Gly-Ala-Gly∼ProMet-Ala-Ala-Arg-Gly paying special attention to the reaction mechanisms involved in these reactive processes, the factors governing the reported selective cleavage, and the effect of the metal and the Pro amino acid. Thus, we performed first QM optimizations on small cluster models representing the first coordination shell of the Pd(II) ion bound to the peptide molecules. From these structures, some molecular mechanics (MM) parameters were derived that allowed us to run classical molecular dynamics (MD) simulations to explore different coordination modes of the peptide substrates to the Pd(II) ion and to obtain more realistic models of the Pd(II)-bound peptide molecules in solution. Subsequently, we extracted large cluster models from representative MD snapshots of the two Pd(II)bound oligopeptides. These models, which include explicit water molecules and the closer residues to the Pd(II) center, were used to study the molecular details of the hydrolysis reaction by means of QM calculations. Computational Details MM Parameterization. Binding between transition metal ions and peptide molecules constitutes a challenging problem for biomolecular modeling because of the ability of these metal ions to assume a variety of coordination states. In this work, we used a mixed bonded and nonbonded representation in which the Pd(II) ion and the four donor atoms placed at the equatorial positions are linked through explicit MM bonds, while the looser Pd-water interactions at the axial positions are represented by nonbonded parameters. We refined first the van der Waals parameters of the Pd(II) ion. Thus, we optimized the square planar [Pd(H2O)4]2+ complex at the B3LYP/6-31G(d) (LANL2DZ for Pd) level of theory63-67 followed by analytical frequency calculations and using the Gaussian 03 series of programs.68 The atomic partial charges were derived by means of the RESP method69 and using the B3LYP/6-31G(d) electrostatic potential. The force constants for the bond (Pd-O) and angle (O-Pd-O, H-O-Pd) MM terms were taken from the frequency calculations, while the torsions associated with the Pd-ligand interactions were set to zero.

Peptide Hydrolysis by a Pd(II) Aqua Complex Subsequently, we solvated the [Pd(H2O)4]2+ system inside a box with a side length of 20 Å containing TIP3P waters. We fixed a van der Waals radius of 1.7 Å for the Pd ion,70 and the well depth (ε) was varied between 0.01 and 0.4 kcal/mol. For each value of ε, a 2 ns MD simulation was carried out, and the radial distribution functions g(r) between the Pd and the O atoms of the TIP3P waters were computed. The corresponding peaks in g(r) were integrated. In this way, we chose ε ) 0.05 kcal/mol because the resulting solvation shell around the Pd(II) ion is in agreement with the published results for the hydration structure of [Pd(H2O)4]2+ as obtained with a more sophisticated potential (this means two axial waters about 2.7 Å from Pd and 10 other water molecules in the second solvation shell).71 We note that another van der Waals parameter for Pd(II) had also been reported in the literature,70,72 but they did not give satisfactory results for the [Pd(H2O)4]2+ complex. Initial geometries (bond lengths and angles) for the Pd coordination sphere in the presence of the oligopeptides were obtained from QM geometry optimizations of small Pd(II)peptide complexes mimicking the first coordination shell of the Pd(II)-peptide complexes. Thus, Ace-Gly∼Gly-Met-Nme and Ace-Gly∼Pro-Met-Nme sequences bound to Pd(II) were built by molecular modeling according to the binding modes 1 and 2 in Scheme 2. For the Ace-Gly∼Gly-Met-Nme peptide, both the cis- and trans-isomer of the Gly∼Gly peptide bond in 1 and the trans one in 2 were constructed. The trans conformation of the Gly∼Pro peptide bond both in 1 and in 2 was only considered for the Ace-Gly∼Pro-Met-Nme peptide. This resulted in five different complexes that were fully optimized at the B3LYP/6-31G(d) (LANL2DZ for Pd) level and characterized by analytical frequency calculations (see Figure S1, Supporting Information). The IEF-PCM continuum solvent model73,74 was applied in these calculations to mimic an organic solvent environment (ε ) 4.0) in accordance with the prescriptions for parameter derivation as described in the AMBER03 protocol.75 From the equilibrium geometries and the normalmode analyses, we obtained all the required reference values and force constants for the bond (Pd-X) and angle (X-Pd-Y) MM terms to represent the coordination modes shown in Scheme 2. The rest of the atoms in the peptide molecules were assigned the corresponding standard AMBER03 bond/angle/ torsion parameters and atom types. The atomic partial charges for the Pd(II) and its ligands were adjusted to the B3LYP/ccpVTZ electrostatic potential using the RESP methodology.69 During the RESP fitting procedure, we imposed the AMBER03 charges for the Ace and Nme residues. All the generated parameters are given in the Supporting Information. MD Simulations of the Pd(II)-oligopeptide Systems. Starting from the optimized geometries in the gas phase of the possible complexation modes between the Ace-Gly∼Gly-MetNme or Ace-Gly∼Pro-Met-Nme tripeptides and the Pd(II) ion, we added the corresponding residues to simulate the full oligopeptides Ace-Ala-Lys-Tyr-Gly∼Gly-Met-Ala-Ala-Arg-Ala and Ace-Lys-Gly-Gly-Ala-Gly∼Pro-Met-Ala-Ala-Arg-Gly that were considered experimentally.20 The pKa values for the Ala and Gly terminal amino acids of the two peptide sequences were calculated using the empirical SPARC method,76 finding that these terminal amino acids must be protonated at pH 2. Hence, we generated the corresponding atomic charges for the Cterminal Ala-COOH and Gly-COOH residues by proceeding as in the previous cases mentioned above. The systems were hydrated with explicit TIP3P water molecules and neutralized with three Cl- counterions. For the

J. Phys. Chem. B, Vol. 114, No. 25, 2010 8527 cis conformation of the complexation mode 1 in the Gly∼GlyMet case, denoted hereafter by 1-Glycis peptide sequence, a box dimension of 60 × 58 × 53 Å3 (7337 water molecules) was assumed in the calculations. For the trans conformations case, box dimensions in the Gly∼Gly-Met sequence were 66 × 59 × 77 Å3 (8244 water molecules) and 65 × 63 × 69 Å3 (7464 water molecules) for the complexation modes 1 and 2, respectively, whereas in the Gly∼Pro-Met case they were 58 × 72 × 77 Å3 (8824 water molecules) and 57 × 70 × 71 Å3 (7453 water molecules) for the complexation modes 1 and 2, respectively. The AMBER03 Duan et al.’s force-field75 was used to model the solvated peptides. Energy minimizations and MD simulations were carried out using the SANDER and PMEMD programs included in the AMBER 9.0 suite of programs.77 To eliminate bad contacts in the initial geometries, we carried out the following cycle: (1) relaxation of the solvent molecules and counterions by means of energy minimizations, (2) energy minimization of the peptides, (3) energy minimizations of the solvent molecules and counterions, (4) 100 ps of MD simulation for relaxing the solvent molecules and the counterions, and (5) energy minimization of the full systems. The SHAKE algorithm78 was employed to constrain all the R-H bonds, in which R is any atom linked to the H atom, and periodic boundary conditions were applied to simulate a continuous system. A cutoff of 10.0 Å was defined for the nonbonded interactions, and the particle-mesh Ewald (PME) method was used to include the long-range contributions.79 Berendsen’s method80 was used to control the pressure (1 atm) and the temperature (300 K) of the system during the MD simulations. A 20 ns trajectory was computed for each model with a time step of 2 fs, but only the last 8.0 ns of each trajectory was analyzed (coordinates were saved for analysis every 500 ps). A set of MD snapshots were grouped in different clusters according to the criterion of a fixed radius of displacement of the backbone with respect to the values of the root-mean-square deviation (RMSD) for the Φ and Ψ backbone angles. The structure in each cluster with the lowest deviation is taken as the cluster representative. Other structural analyses were done using the PTRAJ module of AMBER 9.0. Energetic Analyses of the MD Simulations. To estimate the average free energies for the simulated Gly∼Gly-Met and Gly∼Pro-Met systems, a set of 50 snapshots were extracted every 100 ps from the production phase of each MD trajectory. The coordinates of the water molecules and counterions were removed excepting those of a cap of 25 water molecules centered on the Pd(II) ion. Then, the average free energy of the models was estimated according to the following equation COSMO j ≈E j QM + E j disp + ∆G ¯ solv G

(1)

j is the calculated average free energy; E j QM is the average where G QM energy of the solute and the remaining water molecules; j disp is an empirical energy that takes into account the attractive E j solv is the average solvation dispersive interactions; and ∆G energy, which is calculated using a QM Hamiltonian coupled with a continuum model. The TURBOMOLE V5.9 program81 was used to carry out these single-point energy calculations at the PBE82,83/def2-TZVP84 level of theory with the COSMO model85 to simulate the rest of the water bulk. The dispersion energy contribution, Edisp, was computed using an empirical formula that has been introduced by Elstner et al. to extend

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their approximate DFT method for the description of dispersive interactions, which are normally neglected in the DFT methods.86 QM Study of the Reaction Mechanism. On the basis of the structures generated by the MD simulations, we built cluster models which consisted of the Pd(II) ion, the Gly∼Gly-MetAla and Gly∼Pro-Met-Ala moieties capped with Ace and Nme groups, two Pd-bound water molecules, as well as 15 other waters closest to the Pd ion. The investigation on the reaction mechanism for the resulting cluster models was carried out with the Gaussian 03 package. Full geometry optimizations of stable species and transition states (TS) were performed in the gas phase at the B3LYP/6-31G(d) (LANL2DZ for Pd) level of theory and by using the standard Schlegel’s algorithm.87 The nature of the stationary points found was verified by analytical computations of harmonic vibrational frequencies. Intrinsic reaction coordinate (IRC) calculations with the Gonzalez and Schlegel method88,89 were carried out to check the two minimum energy structures connecting each TS. Thermodynamic magnitudes were computed within the ideal gas, rigid rotor, and harmonic oscillator approximations at a pressure of 1 atm and a temperature of 298.15 K.90 To take into account condensed phase effects, single-point calculations were also performed on the gas phase optimized geometries using a conductor-like polarizable continuum model (CPCM)91,92 with Klamt’s radii (COSMO). To remove the energetic noise in the Gibbs energy barriers of the reaction mechanisms produced by the small differences in the energy contributions of the explicit water molecules, to include the effects of long-range electrostatic interactions, as well as to refine the electronic energies, we performed singlepoint high-level energy calculations on the located critical structures preserving only the waters anchored to the Pd atom and those implied in the reaction coordinate. For accomplishing this task, we employed the RI93-MP2/def2-TZVPP84 theory level as implemented in the TURBOMOLE package to calculate the electronic energy in the gas phase along with the B3LYP/631G(d) theory level to calculate the free energy of solvation by using the COSMO model as implemented in Gaussian 03. Results Molecular Dynamics Simulations: Determination of Hydrolytically Active Complexes. Experimental results on the hydrolysis of Ace-Ala-Lys-Tyr-Gly∼Gly-Met-Ala-Ala-Arg-Ala and Ace-Lys-Gly-Gly-Ala-Gly∼Pro-Met-Ala-Ala-Arg-Gly catalyzed by the Pd(II) ion have pointed out that the binding mode type 1 in Scheme 2, in which the square planar Pd(II) ion is coordinated by two water molecules and the sulfur and nitrogen atoms of the methionine amino acid, is the hydrolytically active complex for both oligopeptides.20 However, experimentalists did not rule out the existence of an additional interaction between the Pd(II) ion and the Gly-carbonyl oxygen of the peptide bond to be cleaved (see complex 2 in Scheme 2), as a consequence of the trans conformation detected for the Gly5-Pro6 bond in the case of the Gly∼Pro-Met peptide.20 Furthermore, for the Gly∼Gly-Met peptide case, a hypothetical cis conformation has been assigned to the scissile peptide bond of the binding mode 1 (see Scheme 2) on the basis of the basicity of the amide carbonyl oxygen atom of this bond.20,94 Since the trans conformation of peptide bonds is normally more stable than the cis one and there is no direct experimental evidence on the cis-trans character of the Gly-Gly linkage in Ace-Ala-LysTyr-Gly∼Gly-Met-Ala-Ala-Arg-Ala, we decided to computationally examine the two isomers using MD simulations.

Yeguas et al. Therefore, with of all of this in mind, our first goal was to determine the geometry and relative energy of the various types of hydrolytically active complexes in aqueous solution. As described in the Computational Details section, we parametrized the Pd(II) coordination environment within the context of the AMBER03 force field. Subsequently, we performed five different MD simulations (1-Gly, 2-Gly, 1-Pro, 2-Pro, and 1-Glycis) of the full peptide sequences in water considering the experimental conditions (pH 2), the two possible Pd coordination modes, and the cis and trans isomers for the Gly system. All the trajectories were started from a conformation resembling a β-hairpin motif with the Pd(II) ion placed nearby the peptide turn. In four of the MD simulations, the initial structures evolved dynamically toward more compact structures as shown by the plots of their radius of gyration (see Figure S2 in the Supporting Information). For example, we observed that the radius of gyration of the 1-Gly model peptide fluctuates widely between 9.7 and 7.0 Å during the first 10 ns, but it becomes largely stabilized in the second half of the trajectory fluctuating smoothly around 6.6 ( 0.2 Å. Similarly, the 1-Pro model adopts a stable conformation characterized by a radius of gyration of 5.8 ( 0.2 Å. The models with a tricoordinated oligopeptide 2-Gly and 2-Pro exhibit a different behavior: 2-Gly leads again to a very stable conformation (rgyr ) 6.4 ( 0.2 Å), whereas the 2-Pro turns out to be quite flexible according to its average rgyr value, 8.1 ( 1.0 Å. To further assess the large mobility of the latter model, the MD simulation of 2-Pro was extended up to 40 ns (data not shown for brevity). On the other hand, the rgyr values of the 1-Glycis system with the Gly∼Gly bond in cis conformation kept fluctuating between 6 and 9 Å, suggesting thus that the simulation was well equilibrated and that the resulting flexibility (rgyr ) 5.8 ( 0.9 Å) is an intrinsic dynamic feature of this system. The superposition of the most important cluster representatives corresponding to the different models is shown in Figure 1. On the basis of their root-mean-square similarity, the representative structures account for ∼85% of the sampled snapshots. According to the clustering analyses, three and four representative structures account for the majority of the MD snapshots of the dicoordinated 1-Gly and 1-Pro models, respectively. In fact, the various structures are quite similar to each other and point out that the largest conformational flexibility arises at the N- and C-terminal residues. Besides the Pd(II)-peptide bonds, the conformation of the peptide molecules is mainly stabilized by direct and/or water-mediated H-bond interactions connecting backbone atoms of the two peptide ends (e.g., Gly5-NH · · · O-Ala8; Arg10-NH · · · O-Ala2, in the 1-Gly model). As expected, the central residues of the oligopeptide molecules, including the Pd(II)-coordinated Met residue, have a low flexibility. However, it is interesting to note that the Pd(II) site in the 1-Pro model is significantly more rigid than in the 1-Gly model. In particular, the root mean squared flexibility (RMSF) values for the backbone atoms in the central residues, Tyr4-Gly5∼Gly6-Met7-Ala8 in 1-Gly and Ala5-Gly6∼Pro7-Met8Ala9 in 1-Pro, are 0.74 ( 0.19 and 0.38 ( 0.14 Å, respectively. This is well understood in terms of the presence of the imidic ring in the Pro7 residue, which imposes important conformational restrictions on the Pro-containing peptide chains.95 For the tricoordinated models, 2-Gly and 2-Pro, a single cluster representative accounting for 80% of the analyzed snapshots describes a very stable 2-Gly conformation, which is characterized by a few intramolecular H-bond interactions (Ace1-CdO · · · · H2Nε-Arg10; Ala2-CdO · · · HN-Arg10). In con-

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Figure 1. Superposition of the most populated representative structures derived from the clustering analyses of the MD simulations. The thickness of the models corresponds to the number of snapshots represented by each model.

Figure 2. Gibbs energy profiles corresponding to the hydrolysis of the peptide Ace-Gly∼Gly-Met-Ala-Nme catalyzed by the Pd(II) ion at the B3LYP/6-31G(d) (LANL2DZ for Pd) level of theory.

trast, many more representative structures are required for 2-Pro that can be seen as structurally disordered excepting the residues in the vicinity of the Pd(II) ion (see Figure 1). The source of the large dynamical variability of 2-Pro could be related to the presence of two extra glycine residues (Gly3-Gly4) in its peptide sequence, which become more solvent accessible when the peptide turn is widened in response to the tricoordination of the peptide molecule with the Pd(II) ion. The conformational variability of the 1-Glycis configuration is described by four structures that account for 70% of the analyzed snapshots; that

is, the flexibility of 1-Glycis is similar to that of 1-Gly. Note again that the positioning of the N- and C-terminal groups is quite variable in the 1-Glycis representatives, although the region close to the Pd center is structurally quite rigid (see Figure 1). The structures generated by the MD simulations can also provide insight into the nature of the hydrolysis mechanism. First, the MD models of the tricoordinated complexes, 2-Gly and 2-Pro, confirm that the Pd(II)-bound water molecule is not well poised for acting as the nucleophile against the carbonyl group of the scissile peptide bond (see Figure 1). In the case of

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Figure 3. Gibbs energy profiles corresponding to the hydrolysis of the peptide Ace-Gly∼Pro-Met-Ala-Nme catalyzed by the Pd(II) ion at the B3LYP/6-31G(d) (LANL2DZ for Pd) level of theory.

the dicoordinated configurations 1-Gly and 1-Pro, one of the Pd(II)-bound water molecules is H-bonded with the O atom of the reactive carbonyl group (Pd-OH2 · · · OdC-Gly ) 2.6 ( 0.2 Å). However, the Pd-bound water and the amide group are nearly coplanar all along the MD trajectories, and consequently, a direct nucleophilic attack of the Pd-bound water molecule is sterically unfavorable. Similarly, in the 1-Glycis configuration, the relative positioning between the scissile Gly∼Gly amide bond and the Pd-bound Wat1/Wat2 molecules is clearly not adequate for promoting an internal Pd-WatfGly5 reactive event (see Figure 1) because the interatomic distance between the O(Wat) and C(Gly5) atoms is always larger than 5 Å during the MD simulations. Therefore, we conclude that the peptide cleavage assisted by the Pd(II) ion must take place through an external attack of a water molecule as originally proposed in experimental studies.20 In this respect, it is interesting to note that the Tyr4 ring in the 2-Gly model reduces the accessibility to the hydrolyzable peptide bond (see Figure 1). Energetic Analyses of the MD Trajectories. In principle, the relative energetic stability of the Pd(II)-peptide configurations is governed by several factors (e.g., the strength of metal-peptide/metal-water bonds; peptide-solvent interactions, etc.) that can be taken into account by means of approximate free energy calculations on a series of MD snapshots. Thus, from the G values in Table S1 (Supporting Information), the relative stability of the dicoordinated and tricoordinated models can be assessed directly. We note first that the 1-Glycis configuration is predicted to be 8.2 kcal/mol less stable than 1-Gly and that this energy difference is clearly larger than its statistical uncertainty (3.1 kcal/mol). Moreover, a similar energy difference resulted (10.2 kcal/mol) in the preliminary DFT calculations on the small cluster models (see Supporting Information), showing thus that neither the Pd(II) nor the rest of the amino acids and water environment modify the general energetic preference for the trans-isomer. For the 1-Pro and 2-Pro models, it turns out that the coordination mode 1 (see Scheme 2) in which the Pd(II) ion is coordinated by the N and S atoms of the Met residue is 15.4 kcal/mol more stable than the tricoordinated mode including the O carbonyl group of the Gly residue. Although the statistical uncertainty (5.2 kcal/mol) is quite large due to the large flexibility of the 2-Pro configuration, the large magnitude of this energy difference allows us to safely assign the 1-Pro model as the most likely configuration of the Pd(II)-peptide complex in solution. On the other hand, the dicoordinated structure 1-Gly is also predicted to be more stable by 1.7 kcal/mol than 2-Gly.

However, we also note that both the 1-Gly and 2-Gly configurations could be energetically accessible as their corresponding energy difference is comparable to the standard error of the mean free energies (∼2 kcal/mol). Moreover, more sophisticated energy calculations and/or inclusion of solute entropic could also affect this small energy difference. Mechanism of the Hydrolysis Reaction. The most populated cluster representative of each trajectory was used as the starting point to investigate the reaction mechanism of the hydrolytic process. Given that the central region of the peptide molecules nearby Pd(II) is conformationally rigid, we truncated the MD snapshots by retaining only the Pd(II) ion, the central residues capped by Ace and Nme groups, and the closest water molecules around the metal atom. The resulting structures still retain the basic information that is necessary to accomplish the study of the hydrolysis mechanism. Figures 2 and 3 collect the Gibbs energy profiles in water solution corresponding to the reaction mechanisms found for the hydrolysis of the peptides Ace-Gly∼Gly-Met-Ala-Nme and Ace-Gly∼Pro-Met-Ala-Nme catalyzed by the Pd(II) ion, respectively, at the B3LYP/6-31G(d) (LANL2DZ for Pd) level of theory. Figures 4 and 5 display the corresponding optimized geometries of the structures involved in the reaction mechanisms found, and Table S2 in the Supporting Information collects their corresponding energies. Unless otherwise stated, we will give in the text the relative Gibbs energies in water solution of all the located species. In the case of the peptide sequence Ace-Gly∼Gly-Met-AlaNme, we focus our attention on the hydrolytic cleavage mechanism of the binding modes 1-Gly and 2-Gly, that is, the trans conformation of binding modes 1 and 2 collected in Scheme 2, respectively. Two different stepwise reaction mechanisms for the complex 1-Gly and one concerted reaction mechanism for 2-Gly were located. Starting from 1-Gly, the most favorable route corresponds to the formation of the diol intermediate I1A-1Gly, which is 18.0 kcal/mol less stable than 1-Gly, and through the TS TS1A-1Gly, 7.3 kcal/mol higher in energy than I1A-1Gly. At TS1A-1Gly, a H atom of one of the two water molecules directly bound to Pd is practically transferred to the carbonyl oxygen atom of the Gly∼Gly amide bond since the distances H · · · O(carbonyl) and H · · · O(Pd) are 1.011 and 1.616 Å, respectively. Simultaneously, an external water molecule, which is interacting with the carbonyl carbon atom of the Gly∼Gly amide bond at a distance of 1.649 Å, increases its nucleophilicity because one of its H atoms exhibits an interaction with another external water molecule, which in



Figure 4. B3LYP/6-31G(d) (LANL2DZ for Pd) optimized geometries of the peptide sequence Ace-Gly∼Gly-Met-Ala-Nme catalyzed by Pd(II).

Figure 5. B3LYP/6-31G(d) (LANL2DZ for Pd) optimized geometries of the peptide sequence Ace-Gly∼Pro-Met-Ala-Nme catalyzed by Pd(II).

turn presents a H atom interacting with the deprotonated oxygen atom linked to Pd at a distance of 1.447 Å. As a consequence, at I1A-1Gly the carbonyl carbon atom of the Gly∼Gly amide bond becomes a tetrahedral carbon atom linked to two hydroxyl groups. The following step is for the protonation of the nitrogen atom of the Gly∼Gly amide bond through the TS TS2A-1Gly, 29.1 kcal/mol less stable than 1-Gly, in which a H atom of another external water molecule is interacting with the N atom of the Gly∼Gly amide bond, while the diol H atom linked to the original first external water molecule is practically transferred to the third external water molecule mentioned above. TS2A1Gly evolves to the intermediate I2A-1Gly, 14.0 kcal/mol lower in energy than the former TS, in which the amidic nitrogen atom is protonated and the amidic C∼N bond is slightly lengthened (1.614 Å). Then, I2A-1Gly undergoes the cleavage of the amidic C∼N bond of the Gly∼Gly amide linkage through the TS

TS3A-1Gly, 15.5 kcal/mol less stable than 1-Gly, to give the preproduct complex P3A-1Gly, 10.1 kcal/mol higher in energy than 1-Gly. The rate-determining step of this reaction mechanism is the second one, which presents a Gibbs energy barrier in solution of 29.1 kcal/mol. The other mechanistic route starting from 1-Gly proceeds through the TS TS1B-1Gly, 44.6 kcal/ mol higher in energy than 1-Gly, for the addition of one of the O-H bonds of an external water molecule to the C∼N bond of the Gly∼Gly amide linkage with simultaneous migration of a H atom of one of the water molecules directly bound to Pd to the amidic oxygen atom. This TS leads to the tetrahedral intermediate I1B-1Gly, 13.3 kcal/mol less stable than 1-Gly, in which the amidic carbon atom is linked to two hydroxyl groups, with the amidic nitrogen atom also being protonated. From here, the system evolves through the TS TS2B-1Gly, 14.4 kcal/mol less stable than 1-Gly, to give the preproduct complex

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P3B-1Gly, 5.6 kcal/mol higher in energy than 1-Gly, for the cleavage of the amidic C∼N bond with a simultaneous H transfer from one of the hydroxyl O atoms linked to the diol C atom to the deprotonated O atom linked to the metal center. The first step of this mechanistic route is the rate-determining one with a Gibbs energy barrier in solution of 44.6 kcal/mol. Finally, the only reaction mechanism located starting from 2-Gly implies solely the TS TS1A-2Gly, 39.3 kcal/mol higher in energy than 2-Gly, wherein two external water molecules interact with the C∼N bond of the Gly∼Gly amide linkage as follows. The oxygen atom and a H atom of one of the water molecules are interacting with the carbonyl carbon atom and the oxygen atom of the other water molecule at distances of 1.584 and 1.149 Å, respectively, while the latter water molecule presents one of its H atoms interacting with the amidic nitrogen atom at a distance of 1.781 Å. The corresponding preproduct complex P1A-2Gly is 21.5 kcal/mol less stable than 2-Gly. Taking into consideration the rate-determining energy barriers of the reaction mechanisms obtained for the Ace-Gly∼GlyMet-Ala-Nme sequence together with the fact that 2-Pro is about 15 kcal/mol less stable than 1-Pro, in the case of the Ace-Gly∼Pro-Met-Ala-Nme sequence, we only investigated the reaction mechanism starting from 1-Pro, which involves the formation of a diol intermediate without amidic N protonation. To reach the diol intermediate I1A-1Pro, which is 19.4 kcal/mol less stable than 1-Pro, the system evolves through the TS TS1A-1Pro, which implies an energy barrier of 26.2 kcal/ mol. TS1A-1Pro is analogous to TS1A-1Gly but presents a slightly higher energy barrier due to a longer distance between the amidic carbonyl carbon atom of the Gly∼Pro bond and the oxygen atom of the external water molecule (1.705 versus 1.649 Å). From I1A-1Pro, the system evolves through the TS TS2A1Pro, 31.6 kcal/mol higher in energy than 1-Pro, to give the intermediate I2A-1Pro, 12.0 kcal/mol lower in energy than TS2A-1Pro. This TS involves again the protonation of the nitrogen amidic atom as in TS2A-1Gly, but the comparative analysis of both TS reveals a notable difference. At TS2A-1Gly, a H atom is practically transferred from one of the hydroxyl groups linked to the amidic carbonyl carbon atom to the external water oxygen atom (the O(CN) · · · H and O(water) · · · H distances are 1.442 and 1.095 Å, respectively), while this H atom is practically in the middle of the distance O(CN) · · · O(water) (the O(CN) · · · H and O(water) · · · H distances are 1.259 and 1.197 Å, respectively) at TS2A-1Pro. Although less pronounced, a similar image was found for the H atom, which is placed between the amidic nitrogen atom and the external water oxygen atom (the N(CN) · · · H and O(water) · · · H distances are 1.589 and 1.060 at TS2A-1Gly, respectively, and 1.350 and 1.184 at TS2A-1Pro, respectively). The more advanced H transfer to the N atom at TS2A-1Pro is in agreement with the more basic character of the Pro amine group with respect to the Gly amine group.20 At I2A-1Pro, the two H atoms are transferred to the corresponding O and N atoms. This intermediate undergoes the cleavage of the Gly∼Pro amidic C∼N bond through the TS TS3A-1Pro, which presents the same relative energy as the previous intermediate, to give the preproduct complex P3A1Pro, 10.7 kcal/mol lower in energy than I2A-1Pro. The comparison of TS3A-1Pro with the analogous one for the AceGly∼Gly-Met-Ala-Nme case, TS3A-1Gly, reflects that the amidic C∼N distance is notably more shortened (1.034 Å) at the former TS than at the latter one. As for 1-Gly, the second step of this mechanistic route is again the rate-determining one with a Gibbs energy barrier in solution of 31.6 kcal/mol.

Yeguas et al. Discussion and Comparison with Experiment The most stable configurations 1-Gly and 1-Pro predicted by our MD and QM calculations show their corresponding scissile Gly-Gly and Gly-Pro peptide bonds in trans conformation. This computational result confirms the hydrolytically active complex experimentally proposed for the Gly∼Pro-Met sequence on the basis of NMR data but is in contrast to the suggestion of a possible cis conformation of the scissile peptide bond for the Gly∼Gly-Met sequence.20 Besides this, it is interesting to remark how the MD analyses have revealed the presence of an interaction between one of the hydrogen atoms of one of the equatorial water molecules linked to the Pd ion and the carbonyl oxygen atom involved in the amidic C∼N bond to be cleaved. Other interesting points can be deduced both for the conformation of the hydrolytically active complex and for the reaction mechanism of hydrolysis. On one hand, the analysis of the MD structures and the QM critical points involved in the reaction mechanisms shows that the Pd anchorage to the S and N atoms of the Met residue forces the peptide backbone to adopt a hairpin-like shape, thus explaining the cleavage of the second peptide bond upstream as experimentally reported.20 On the other hand, our results also coincide with the experimental suggestions, which indicate that the hydrolytic process takes place through an external attack of a water molecule to a trans conformer (see 2 in Scheme 2).20 However, our calculations indicate that the carbonyl oxygen atom involved in the amidic bond cleavage is interacting with a Pd-coordinated water molecule instead of directly bound to Pd as experimentally suggested.20 Coordination to a metal cation weakens the nucleophilicity of this water molecule. On one hand, this weakening activates the carbonyl bond involved in the amidic bond cleavage thanks to the proton transfer from this water molecule to the carbonyl oxygen atom, thus facilitating the attack of the external molecule water. On the other hand, although that weakening could disfavor the hydrogen bonding between the oxygen atom of that Pd-coordinated water molecule and a hydrogen atom of the external water molecule directly linked to it, our results indicate the establishment of this interaction at the first step of the mechanism of hydrolysis reaction (see Figures 4 and 5). In accordance with our theoretical results on the hydrolysis of the sequences Ace-Gly∼Gly-Met-Ala-Nme and AceGly∼Pro-Met-Ala-Nme catalyzed by Pd(II), the most favorable reaction mechanism starts with the formation of a diol intermediate, which subsequently proceeds through the protonation of the amidic nitrogen atom of the Gly∼Gly and Gly∼Pro linkages and then evolves for the cleavage of the C∼N bond. For both sequences, the rate-determining step corresponds to the second one, which presents a Gibbs energy barrier in solution of 29.1 and 31.6 kcal/mol for the Gly∼Gly-Met and Gly∼ProMet fragments, respectively. This indicates a higher reactivity of the Gly case over the Pro one, which is in contrast to the experimental evidence that Pro is more reactive than Gly.20 However, based on the rate constants experimentally reported at pH 2 for the [Pd(H2O)4]2+-catalyzed hydrolysis of the synthetic peptides Ace-Ala-Lys-Tyr-Gly∼Gly-Met-Ala-AlaArg-Ala and Ace-Lys-Gly-Gly-Ala-Gly∼Pro-Met-Ala-AlaArg-Gly, the activation Gibbs energies of the former hydrolysis would only be about 1.8 kcal/mol higher in energy than that of the latter one by using the classical Transition State Theory equation. This small value is the range of the accuracy of the most high-level quantum-chemical calculations. Therefore, to accomplish this task, we focused our attention on the energy of the species involved in the most favorable reaction mechanism



TABLE 1: RI-MP2/def2-TZVPP Relative Electronic Energies in the Gas Phase (∆E), B3LYP/6-31G(d) (LANL2DZ for Pd) Gibbs Energies of Solvation (∆Gsolvation and ∆∆Gsolvation), and Relative Gibbs Energies in Water Solution (∆Gsol) for the Critical Structures Located along the Most Favorable Reaction Mechanisms Found for the Pd(II)-Promoted Hydrolysis of the Sequences Ace-Gly∼Gly-Met-Ala-Nme and Ace-Gly∼Pro-Met-Ala-Nme by Taking into Account Only the Water Molecules Directly Implied in the Reaction Coordinatea species

∆E

∆Gsolvation

∆∆Gsolvation

∆Gsol

1-Gly TS1A-1Gly I1A-1Gly TS2A-1Gly I2A-1Gly TS3A-1Gly PA-1Gly 1-Pro TS1A-1Pro I1A-1Pro TS2A-1Pro I2A-1Pro TS3A-1Pro PA-1Pro

0.0 16.3 6.5 23.6 8.1 13.2 8.2 0.0 19.8 20.0 24.8 11.2 19.8 -5.9

-44.5 -42.6 -43.0 -43.8 -46.3 -44.9 -46.2 -39.4 -37.5 -38.6 -40.6 -41.4 -42.3 -41.7

0.0 1.9 1.5 0.7 -1.7 -0.4 -1.7 0.0 1.9 0.8 -1.2 -2.0 -2.9 -2.3

0.0 18.2 8.0 24.3 6.4 12.8 6.5 0.0 21.7 20.8 23.6 9.2 16.9 -8.2

a

All the magnitudes are given in kcal/mol.

for both peptide sequences by performing more sophisticated single-point energy calculations on the gas phase geometries at the RI-MP2/def2-TZVPP level of theory (see Table 1). We wish to remark here that these very demanding calculations were carried out by taking only the two water molecules directly linked to Pd(II) and the three molecules involved in the reactive process, instead of the 15 water molecules initially considered. As can be seen in Table 1, the second step is again the ratedetermining one for both cases, but now the hydrolysis of the Gly∼Pro bond in the Gly∼Pro-Met sequence presents a ratedetermining energy barrier in solution 0.7 kcal/mol lower in energy than that of the Gly∼Gly bond in the Gly∼Gly-Met sequence. In addition to the observed differences in the free energy barriers for the cluster models of the Gly∼Gly-Met and Gly∼Pro-Met systems, it may be interesting to note that the equilibrium and dynamical properties of the fully solvated Pd(II)-peptide complexes can also contribute to modulate their kinetic behavior. For example, the abundance of the prereactive complexes in the Gly∼Gly-Met peptide might be reduced with respect to Gly∼Pro-Met due to a possible equilibrium in aqueous solution connecting the dicoordinated (1-Gly, reactive) and the tricoordinated (2-Gly, no reactive) structures. Our approximate free energy calculations indicate that 1-Gly is 1.7 kcal/mol more stable and thereby would be the predominant form (94% abundance), but the actual population value could be slightly modified by more accurate energy calculations. More interestingly, it is the fact that, from the viewpoint of the hydrolysis reaction mechanism, the existence of a chain of water molecules connecting the hydrolytic water with the Pdcoordinated water is necessary, which protonates the Gly carbonylic oxygen atom (see Scheme 3). We found that such Pd-OH2 · · · (H2O) · · · (H2O)nuc · · · C-Gly association is present in 2.8% and 7.4% of the MD snapshots during the last 7 ns of the 1-Gly and 1-Pro MD trajectories, respectively. Of course, the different population of the water bridges required for the hydrolysis reaction is in consonance with the larger mobility of the central residues in the 1-Gly system. The free energy of water bridge formation (∆Gbridge) can be directly calculated from

SCHEME 3

the probability (P) of bridge formation (∆Gbridge) -RT ln P), and therefore, an estimation of the relative population of the water bridge in terms of free energy means a kinetic effect of about 0.6 kcal/mol favoring the hydrolysis of the Gly∼ProMet peptide. By combining this free energy contribution with the more accurate RI-MP2 energy values mentioned above, the predicted kinetic preference for the hydrolysis of the Gly∼ProMet sequence amounts to ∼1.3 kcal/mol. This value compares reasonably well with that of 1.8 kcal/mol obtained from the experimental kinetic constants as previously indicated.20 Conclusions A computational study on the structure and the hydrolytic cleavage of the peptide sequences Ace-Ala-Lys-Tyr-Gly∼GlyMet-Ala-Ala-Arg-Ala and Ace-Lys-Gly-Gly-Ala-Gly∼ProMet-Ala-Ala-Arg-Gly in complex with one Pd(II) ion confirms that the hydrolytic process starts with an external attack of a water molecule to a complex in trans conformation of the scissile Gly∼Gly and Gly∼Pro peptide bonds as experimentally found for the Gly∼Pro-Met peptide sequence but uncovers the important role played by a water molecule anchored to the Pd atom, which simultaneously interacts with the carbonyl oxygen atom of the scissile peptide bond. Besides, the anchorage of Pd forces the peptide segment to adopt a hairpin shape, thus favoring the cleavage of the second peptide bond upstream from the anchored Met. Finally, the present study reveals for the first time the structures involved in its reaction mechanism. For the two peptide sequences, the most favorable reaction pathway starts with the formation of a diol intermediate, which subsequently evolves through the protonation of the amidic nitrogen atom of the Gly∼Gly and Gly∼Pro bonds and then undergoes the cleavage of the C∼N bond. According to the highest theory level employed by us, our results reproduce well the higher reactivity of the Gly∼Pro-Met fragment over the Gly∼GlyMet one as experimentally found. Therefore, our theoretical results provide valuable information to get a better understanding of these hydrolytic processes and could also be of interest in designing new Pd(II) complexes for the regioselective cleavage of peptides and proteins. Acknowledgment. Financial support from the Ministerio de Ciencia e Innovacioń of Spain and the European Social Fund (project and grant CTQ2004-06309, and project CTQ200763266) is highly appreciated. Supporting Information Available: MM parameters not included in the AMBER force field and charges used in MD simulations for the complexation modes 1 and 2 between Pd(II) and the synthetic peptides. Table S1, Average energies of the complexation modes 1 and 2; Table S2, B3LYP/6-31G(d) (LANL2DZ for Pd) energy data of the species involved in the reaction mechanisms investigated in this work; Figure S1,

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B3LYP/6-31G(d) (LANL2DZ for Pd) optimized geometries of the complexation modes between Pd(II) and the small peptide models; Figure S2, Radius of gyration along the MD simulations. This material is available free of charge via the Internet at http://pubs.acs.org. References and Notes (1) Croft, L. R. Handbook of Protein Sequence Analysis; Wiley: Chichester, UK, 1980. (2) New Methods in Peptide Mapping for Characterization of Proteins; Hancock, W. S., Ed.; CRC Press: Bocat Raton, FL, 1996. (3) Cheng, X.; Shaltiel, S.; Taylor, S. A. Biochemistry 1998, 37, 14005. (4) Baichoo, N.; Heyduk, T. Protein Sci. 1999, 8, 518. (5) Thorner, J.; Emr, S. D.; Abelson, J. N. Applications of Chimeric Genes and Hybrid Proteins, Part A: Gene Expression and Protien Purification; Academic Press: New York, 2000; Vol. 326. (6) Thomas, J. J.; Bakhtiar, R.; Siuzdak, G. Acc. Chem. Res. 2000, 33, 179. (7) Wallace, C. J. A. Protein Engineering by Semisynthesis; CRC: FL, 2000. (8) Proteolytic Enzymes; Beynon, R., Bond, J. S., Eds.; Oxford University: New York, 2001. (9) Heyduk, T.; Baichoo, N.; Heyduk, E. Metal Ions Biol. Syst. 2001, 38, 255. (10) Hubbard, S.; Beynon, R. J. Proteolysis of NatiVe Proteins as Structural Probe; Oxford University: New York, 2001. (11) Colland, F.; Fujita, N.; Ishihama, A.; Kolb, A. Genes Cells 2002, 7, 233. (12) Loizos, N. Method Mol. Biol. 2004, 261, 199. (13) Radzicka, A.; Wolfendsen, R. J. Am. Chem. Soc. 1996, 118, 6105. (14) Jeon, J. W.; Son, S. J.; Yoo, C. E.; Hong, I. S.; Suh, J. Bioorg. Med. Chem. 2003, 11, 2901. (15) Kassai, M.; Ravi, R. G.; Shealy, S. J.; Grant, K. B. Inorg. Chem. 2004, 43, 6130. (16) Chae, P. S.; Kim, M.-S.; Jeung, C.-S.; Lee, S. D.; Park, H.; Lee, S.; Suh, J. J. Am. Chem. Soc. 2005, 127, 2396. (17) Grant, K. B.; Kassai, M. Curr. Org. Chem. 2006, 10, 1035. (18) Deutscher, M. Methods in Enzymology; Academic Press: New York, 1990; Vol. 182. (19) Walker, J. M. The Protein Protocols Handbook; Humana Press: Totowa, NJ, 2002. (20) Milovic, N. M.; Kostic, N. M. J. Am. Chem. Soc. 2003, 125, 781. (21) Rana, T. M.; Meares, C. F. J. Am. Chem. Soc. 1990, 112, 2457. (22) Cremo, C. R.; Loo, J. A.; Edmonds, C. G.; Hatlelid, K. M. Biochemistry 1992, 31, 491. (23) Bar Shimon, M.; Goldshleger, R.; Karlish, S. J. D. J. Biol. Chem. 1998, 273, 34190. (24) Shrivastava, H. Y.; Unni, N. B. Biochem. Biophys. Res. Commun. 2000, 279, 980. (25) Kumar, C. V.; Brunaprapuk, A.; Cho, A.; Chaudhari, A. J. Chem. Soc., Chem. Commun. 2000, 597. (26) Takarada, T.; Yashiro, M.; Komiyama, M. Chem.sEur. J. 2000, 6, 3906. (27) Allen, G. Metal Ions Biol. Syst. 2001, 38, 197. (28) Buckingham, D. A.; Clark, C. R. Metal Ions Biol. Syst. 2001, 38, 43. (29) Datwyler, S. A.; Meares, C. F. Metal Ions Biol. Syst. 2001, 38, 213. (30) Kito, M.; Urade, M. Metal Ions Biol. Syst. 2001, 38, 187. (31) Komiyama, M. Metal Ions Biol. Syst. 2001, 38, 25. (32) Polzin, G. M.; Burstyn, J. N. Metal Ions Biol. Syst. 2001, 38, 103. (33) Manka, S.; Becker, F.; Hohage, O.; Sheldrick, W. S. J. Inorg. Biochem. 2004, 98, 1947. (34) Erxleben, A. Inorg. Chem. 2005, 44, 1082. (35) Zhu, L.; Kostic, N. M. Inorg. Chem. 1992, 31, 3994. (36) Zhu, L.; Kostic, N. M. J. Am. Chem. Soc. 1993, 115, 4566. (37) Zhu, L.; Qin, L.; Parac, T. N.; Kostic, N. M. J. Am. Chem. Soc. 1994, 116, 5218. (38) Parac, T. N.; Kostic, N. M. J. Am. Chem. Soc. 1996, 118, 5946. (39) Parac, T. N.; Kostic, N. M. J. Am. Chem. Soc. 1996, 118, 51. (40) Milinkovic, S. U.; Parac, T. N.; Djuran, M. I.; Kostic, N. M. J. Chem. Soc., Dalton Trans. 1997, 2771. (41) Karet, G. B.; Kostic, N. M. Inorg. Chem. 1998, 37, 1021. (42) Parac, T. N.; Kostic, N. M. Inorg. Chem. 1998, 37, 2141. (43) Parac, T. N.; Ullmann, G. M.; Kostic, N. M. J. Am. Chem. Soc. 1999, 121, 3127. (44) Kaminskaia, N. V.; Ullmann, G. M.; Fulton, D. B.; Kostic, N. M. Inorg. Chem. 2000, 39, 5004. (45) Kaminskaia, N. V.; Kostic, N. M. Inorg. Chem. 2001, 40, 2368. (46) Milovic, N. M.; Kostic, N. M. J. Am. Chem. Soc. 2002, 124, 4759. (47) Zhu, L.; Kostic, N. M. Inorg. Chim. Acta 2002, 339, 104.

Yeguas et al. (48) Milovic, N. M.; Dutca, L. M.; Kostic, N. M. Inorg. Chem. 2003, 42, 4036. (49) Milovic, N. M.; Dutca, L. M.; Kostic, N. M. Chem.sEur. J. 2003, 9, 5097. (50) Sun, X. J.; Zhang, L.; Zhang, Y.; Yang, G. S.; Guo, Z. J.; Zhu, L. New J. Chem. 2003, 27, 818. (51) Johnson, T. W.; Kostic, N. M. J. Serb. Chem. Soc. 2004, 69, 887. (52) Milovic, N. M.; Badjic, J. D.; Kostic, N. M. J. Am. Chem. Soc. 2004, 126, 696. (53) Manka, S.; Becker, F.; Hohage, O.; Sheldrick, W. S. Inorg. Biochem. 2004, 98, 1947. (54) Yang, G.; Miao, R.; Li, Y.; Hong, J.; Zhao, C.; Guo, Z.; Zhu, L. J. Chem. Soc., Dalton Trans. 2005, 1613. (55) Stoffregen, S. A.; Griffin, A. K. K.; Kostic, N. M. Inorg. Chem. 2005, 44, 8899. (56) Dutca, L. M.; Ko, K. S.; Pohl, N.; Kostic, N. M. Inorg. Chem. 2005, 44, 5141. (57) Johnson, T. W.; Kostic, N. M. J. Serb. Chem. Soc. 2006, 69, 887. (58) Zirkovic, M. D.; Rajkovic, S.; Rychlewka, O.; Warzajtis, B.; Djuran, M. I. Polyhedron 2007, 26, 1541. (59) Milovic, N. M.; Kostic, N. M. Metal Ions Biol. Syst. 2001, 38, 145. (60) Milovic, N. M.; Kostic, N. M. Inorg. Chem. 2002, 41, 7053. (61) Sun, X.; Zhang, L.; Zhang, Y.; Yang, G.; Guo, Z.; Zhu, L. New J. Chem. 2003, 27, 818. (62) Matsubara, T.; Hirao, K. Organometallics 2001, 20, 5056. (63) Becke, A. D. Phys. ReV. A 1988, 38, 3098. (64) Becke, A. D. J. Chem. Phys. 1993, 98, 5648. (65) Lee, C.; Yang, W.; Parr, R. G. Phys. ReV. B 1988, 37, 785. (66) Hehre, W. J.; Radom, L.; Pople, J. A.; Schleyer, P. V. R. Ab Initio Molecular Orbital Theory; Wiley: New York, 1986. (67) Hay, P. J.; Wadt, W. R. J. Chem. Phys. 1985, 82, 299. (68) Frisch, M. J.; Trucks, G. W.; Schlegel, H. B.; Scuseria, G. E.; Robb, M. A.; Cheeseman, J. R.; Montgomery, J. A., Jr.; Vreven, T.; Kudin, K. N.; Burant, J. C.; Millam, J. M.; Iyengar, S. S.; Tomasi, J.; Barone, V.; Mennucci, B.; Cossi, M.; Scalmani, G.; Rega, N.; Petersson, G. A.; Nakatsuji, H.; Hada, M.; Ehara, M.; Toyota, K.; Fukuda, R.; Hasegawa, J.; Ishida, M.; Nakajima, T.; Honda, Y.; Kitao, O.; Nakai, H.; Klene, M.; Li, X.; Knox, J. E.; Hratchian, H. P.; Cross, J. B.; Bakken, V.; Adamo, C.; Jaramillo, J.; Gomperts, R.; Stratmann, R. E.; Yazyev, O.; Austin, A. J.; Cammi, R.; Pomelli, C.; Ochterski, J. W.; Ayala, P. Y.; Morokuma, K.; Voth, G. A.; Salvador, P.; Dannenberg, J. J.; Zakrzewski, V. G.; Dapprich, S.; Daniels, A. D.; Strain, M. C.; Farkas, O.; Malick, D. K.; Rabuck, A. D.; Raghavachari, K.; Foresman, J. B.; Ortiz, J. V.; Cui, Q.; Baboul, A. G.; Clifford, S.; Cioslowski, J.; Stefanov, B. B.; Liu, G.; Liashenko, A.; Piskorz, P.; Komaromi, I.; Martin, R. L.; Fox, D. J.; Keith, T.; Al-Laham, M. A.; Peng, C. Y.; Nanayakkara, A.; Challacombe, M.; Gill, P. M. W.; Johnson, B.; Chen, W.; Wong, M. W.; Gonzalez, C.; Pople, J. A. Gaussian 03, revision C.02; Gaussian, Inc.: Wallingford, CT, 2004. (69) Bayly, C. I.; Cieplak, P.; Cornell, W.; Kollman, P. A. J. Phys. Chem. 1993, 97, 10269. (70) Hambley, T. W. Inorg. Chem. 1998, 37, 3767. (71) Martinez, J. M.; Torrico, F.; Pappalardo, R. R.; Sanchez Marcos, E. J. Phys. Chem. B 2004, 108, 15851. (72) Calligaris, M.; Zangrando, E.; Milani, B.; Marson, A. Eur. J. Inorg. Chem. 2005, 704. (73) Tomasi, J.; Mennucci, B.; Cançe`s, E. J. Mol. Struct. (Theochem) 1999, 464, 211. (74) Pomelli, C. S.; Tomasi, J.; Barone, V. Theor. Chem. Acc. 2001, 105, 446. (75) Duan, Y.; Wu, C.; Chowdhury, S.; Lee, M. C.; Xiong, G.; Zhang, W.; Yang, R.; Cieplak, P.; Luo, R.; Lee, T.; Caldwell, J.; Wang, J.; Kollman, P. J. Comput. Chem. 2003, 24, 1999. (76) Hilal, S. H.; Karickhoff, S. W.; Carreira, L. A. Quant. Struct.-Act. Relationships 1995, 14, 348. (77) Case, D. A.; Darden, T. A.; Cheatham, T. E., III; Simmerling, C. L.; Wang, J.; Duke, R. E.; Luo, R.; Merz, K. M.; Pearlman, D. A.; Crowley, M.; Walker, R. C.; Zhang, W.; Wang, B.; Hayik, S.; Roitberg, A.; Seabra, G.; Wong, K. F.; Paesani, F.; Wu, X.; Brozell, S.; Tsui, V.; Gohlke, H.; Yang, L.; Tan, C.; Mongan, J.; Hornak, V.; Cui, G.; Beroza, P.; Mathews, D. H.; Schafmeister, C.; Ross, W. S.; Kollman, P. A. AMBER 9; University of California: San Francisco, 2006. (78) van Gunsteren, W. F.; Berendsen, H. J. C. Mol. Phys. 1977, 34, 1311. (79) Essmann, U.; Perera, L.; Berkowitz, M. L.; Darden, T.; Lee, H.; Pedersen, L. G. J. Chem. Phys. 1995, 103, 8577. (80) Berendsen, H. J. C.; Postma, J. P. M.; van Gunsteren, W. F.; DiNola, A.; Haak, J. R. J. Chem. Phys. 1984, 81, 3684. (81) Ahlrichs, R.; Ba¨r, M.; Baron, H. P.; Ru¨diger, B.; Bo¨cker, S.; Crawford, N.; Deglmann, P.; Ehrig, M.; Eichkorn, K.; Elliot, S.; Furche, F.; Haase, F.; Ha¨ser, M.; Horn, H.; Ha¨ttig, C.; Huber, C.; Huniar, U.; Kattannek, M.; Ko¨hn, A.; Ko¨lmel, C.; Kollwitz, M.; May, K.; Nava, P.; ¨ hm, H.; Patzelt, H.; Rappoport, D.; Rubner, O.; Scha¨fer, Ochsenfeld, C.; O

Peptide Hydrolysis by a Pd(II) Aqua Complex A.; Schneider, U.; Sierka, M.; Treutler, O.; Unterreiner, B.; von Arnim, M.; Weigend, F.; Weis, P.; Weiss, H. Turbomole, 5.9th ed.; Karlsruhe: Germany, 2005. (82) Perdew, J. P.; Burke, K.; Ernzerhof, M. Phys. ReV. Lett. 1996, 77, 3865. (83) Perdew, J. P.; Burke, K.; Ernzerhof, M. Phys. ReV. Lett. 1997, 78, 1396 LP. (84) Weigend, F.; Ahlrichs, R. Phys. Chem. Chem. Phys. 2005, 7, 3297. (85) Klamt, A.; Schuü¨rmann, G. J. Chem. Soc., Perkin Trans. 2 1993, 799. (86) Elstner, M.; Hobza, P.; Frauenheim, T.; Suhai, S.; Kaxiras, E. J. Chem. Phys. 2001, 114, 5149. (87) Schlegel, H. B. J. Comput. Chem. 1982, 3, 214.

J. Phys. Chem. B, Vol. 114, No. 25, 2010 8535 (88) Gonzalez, C.; Schlegel, H. B. J. Chem. Phys. 1989, 90, 2154. (89) Gonzalez, C.; Schlegel, H. B. J. Phys. Chem. 1990, 94, 5523. (90) McQuarrie, D. A. Statistical Mechanics; Harper and Row: New York, 1976. (91) Barone, V.; Cossi, M. J. Phys. Chem. A 1998, 102, 1995. (92) Cossi, M.; Rega, N.; Scalmani, G.; Barone, V. J. Comput. Chem. 2003, 24, 669. (93) Weigend, F.; Ha¨ser, M. Theor. Chem. Acc. 1997, 97, 331. (94) Martin, R. B. Met. Ions Biol. Syst. 2001, 38, 1. (95) Vanhoof, G.; Goossens, F.; De Meester, I.; Hendriks, D.; Scharpe´, S. FASEB J. 1995, 9, 736.

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