Theoretical Study of the Catalytic Mechanism and Metal-Ion

The reaction pathway of deformylation catalyzed by E. coli peptide deformylase (PDF) has been investigated .... These results are consistent with Gold...
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J. Phys. Chem. B 2007, 111, 6236-6244

Theoretical Study of the Catalytic Mechanism and Metal-Ion Dependence of Peptide Deformylase Xian-Hui Wu,†,‡ Jun-Min Quan,*,† and Yun-Dong Wu*,†,§ Laboratory of Chemical Genomics, Shenzhen Graduate School of Peking UniVersity, Shenzhen, China, The School of Life Science, Wuhan UniVersity, Wuhan, China, and Department of Chemistry, The Hong Kong UniVersity of Science and Technology, Clear Water Bay, Kowloon, Hong Kong, China ReceiVed: December 15, 2006; In Final Form: March 23, 2007

The reaction pathway of deformylation catalyzed by E. coli peptide deformylase (PDF) has been investigated by the density functional theory method of PBE1PBE on a small model and by a two-layer ONIOM method on a realistic protein model. The deformylation proceeds in sequential steps involving nucleophilic addition of metal-coordinated water/hydroxide to the carbonyl carbon of the formyl group, proton transfer, and cleavage of the C-N bond. The first step is rate-determining for the deformylation, which occurs through a pentacoordinated metal center. The estimated activation energies with the ONIOM method are about 23.0, 15.0, and 14.9 kcal/mol for Zn-, Ni-, and Fe-PDFs, respectively. These calculated barriers are in close agreement with experimental observations. Our results demonstrate that the preference for metal coordination geometry exerts a significant influence on the catalytic activity of PDFs by affecting the activation of the carbonyl group of the substrate, the deprotonation of the metal-coordinated water, and the stabilization of the transition state. This preference for coordination geometry is mainly determined by the ligand environment and the intrinsic electronic structures of the metal center in the active site of the PDFs

1. Introduction In eubacteria, as well as mitochondria and chloroplasts, protein synthesis is normally initiated by the N-formylated methionine residue, but the maturation of protein requires the removal of the N-formylated methionine residue. The N-formyl group is removed by peptide deformylase (PDF), and the methionine residue is then hydrolyzed by methionine aminopeptidase (MAP).1 The deformylation process is essential for cell growth in bacteria and fungi and is also unique to eubacteria. It does not occur in eukaryotic or archaebacteria protein synthesis,2 making peptide deformylase an attractive target for the design of new antibiotics.3 Previous biochemical and structural studies have revealed that peptide deformylase has a typical zinc-binding motif, HEXXH, found in many zinc metalloenzymes.4,5 This led to the suggestion that peptide deformylase also belongs to the zinc(II) enzyme family. However, recent evidence has shown that the metal in the active site of wild-type E. coli PDF is iron and that zincbinding deformylase (Zn-PDF) is inactive or dramatically less active than nickel- and iron-binding deformylases (Ni-PDF, Fe-PDF),6 even though their native structures are nearly identical. This led to active studies through experimentation and theoretical calculations on the mechanism of this enzyme to find an explanation for the metal-ion dependence of enzymatic activity.7,8 On the basis of a comparison of the crystal structures of Zn-PDF, Ni-PDF, and Fe-PDF bound to Met-Ser-Ala, Becker and co-workers7 found that there are no differences in the overall folds and only minor differences in the active sites. However, Zn was found to bind more tightly than the other * To whom correspondence should be addressed. E-mail: quanjm@ szpku.edu.cn (J.-M.Q.), [email protected] (Y.-D.W.). † Shenzhen Graduate School of Peking University. ‡ Wuhan University. § The Hong Kong University of Science and Technology.

two metals. They proposed that the enormous difference in the catalytic activities of different metal forms of PDF resulted from the tighter binding of the Zn2+ ion as compared to Ni2+ and Fe2+, which would inhibit the transition from a tetrahedral to a pentacoordinated metal center in the transition state. Recent high-resolution X-ray structures of Zn-PDF and Fe-PDF with formate bound to the metal center obtained by Chan and coworkers8a have revealed different binding modes of formate with metal centers. Zn2+ prefers tetracoordination rather than pentacoordination, whereas Fe2+ has a stronger preference for pentacoordination. Chan et al. proposed that the different metal coordination preferences are the key factor in the metaldependent activity of PDF. On the basis of studies on model systems that mimic active-site features, Goldberg and coworkers8b proposed a similar model in which the inherent geometric preference of Fe2+ for a high coordination number accounts for the much higher reactivity of Fe-PDF compared to Zn-PDF. All of these models provided insight into the metalion dependence of the PDF activity, but the detailed catalytic mechanism and the inherent origin of the metal-ion dependence of PDF activity requires further elucidation. For example, how does the coordination geometry preference of the metal center affect the catalytic activity, and what factors control the coordination geometry preference of the metal center? More recently, Russo et al. reported a density functional theory (DFT) study8c on some active site models that include first-shell metal ligands in an attempt to address the metal-ion dependence of PDF. Interestingly, their calculations did not show an obvious metal-ion dependence on activity, with all of the metal forms (Zn2+, Ni2+, Fe2+) having similar activities in terms of the hydrolysis of the formyl peptide bond. This suggests that the small models comprising only the first-shell metal ligands and the simple substrate model might be insufficient to model the reaction. Further investigation is necessary to characterize the

10.1021/jp068611m CCC: $37.00 © 2007 American Chemical Society Published on Web 05/11/2007

Catalytic Mechanism of Peptide Deformylase

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CHART 1: Calculation Models

detailed hydrolysis mechanism and to understand the metalion dependence of the reactivity of PDFs in a full protein environment. Currently, the state-of-the-art method for this purpose is the combined quantum mechanics/molecular mechanics (QM/MM) method,9 which has been widely applied to the study of reaction mechanisms of enzymatic catalysis.10 In the present study, we report combined QM/MM (ONIOM) calculations on the full reaction pathway of deformylation catalyzed by peptide deformylase. The models include the intact enzyme with different metal ions and substrate formyl-MAS (fo-Met-Ala-Ser), with the active site treated by quantum mechanics (QM) and the rest by molecular mechanics (MM) (details given in the Materials and Methods section). For comparison, we also investigated the initial nucleophilic attack of the catalytic hydrolysis reaction on a truncated active site model using density functional theory (DFT). We aimed to characterize the detailed reaction mechanism in the specific enzyme environment, to elucidate the metal-ion dependence of the reactivity, and to analyze the energetic and structural features of the active species along the reaction pathway. 2. Materials and Methods 2.1. Small Model. The truncated active site includes the metal ion; the protein ligands His132, His136, and Cys90; and the amide backbone of Leu91, mimicked by two imidazoles and -S(CH ) -CONH (Chart 1, middle). In addition, the model 2 2 2 also includes metal-bound hydroxide. The calculations with the DFT model were performed using PBE1PBE functionals.11 The 6-31G* basis set was chosen for the C, H, O, and N atoms, and the 6-31+G* and SDD pseudopotential12 were used for the S and metal ions, respectively. The geometry was fully optimized without geometric constraints, and frequency calculations were performed on the same level to evaluate the stationary points. All DFT calculations were performed with the Gaussian 03 package.13 2.2. QM/MM Models. A. Setup of the System. The computational models were derived from the recent X-ray structure of Ni-PDF complexed with the product MAS (Met-Ala-Ser) tripeptide (PDB code 1BS6).7 The initial substrate-enzyme complex was modeled by adding a formyl group at the N-terminal of methionine and replacing the second coordinated water (W2) by the carbonyl oxygen of the formyl group. This modeling slightly changed the initial binding mode of the MAS tripeptide in the enzyme. The missing residues and hydrogen atoms in the primitive structure were added with the SwissPdb Viewer.14 The Zn2+ and Fe2+ forms of the models were created by replacing the Ni2+ ion with Zn2+ and Fe2+, respectively. The model system was composed of a metal ion, two imidazole rings of His132 and His136, the thiolate of Cys90, the coordinated water molecule, the carboxyl group of Glu133, the amide backbone of Leu91, the HCONHCH part of the substrate (Chart 1, right), and the link atoms, with a total of 47

atoms The real system was treated as the molecular mechanics (MM) region. B. QM/MM Calculations. All calculations were performed by the two-layered ONIOM method15 as implemented in the Gaussian 03 program.13 The model system was treated with the density functional theory of the PBE1PBE functional,11 employing the 6-31G* basis set on C, H, O, and N and the 6-31+G* basis set on S. The metals were described by the SDD pseudopotential.12 The real system was treated with the AMBER all-atom force field.16 According to the previous ab initio calculations8c and our own model calculations (Figure S1, Supporting Information), the high-spin states of Fe2+ and Ni2+ are more stable in the active site of deformylase. Therefore, our calculations treat only the high-spin states of Ni-PDF and Fe-PDF. Because the formation of the Cδ-Oω and Hω-Nδ bonds (Chart 1) comprises two critical steps in the deformylation process, we chose Cδ-Oω and Hω1-Nδ as the reaction coordinates (RCs). An iterative restrained optimization procedure was then repeatedly applied to different points along the reaction coordinates, resulting in a minimum-energy path. For the obtained stationary points, Hessian matrixes were calculated at the same level as the optimization, and the corresponding vibrational frequencies were determined. The energy maximum on the path with only one imaginary frequency was located as the transition state. The Wiberg bond index17 for the metal-Oδ bond was calculated according to Reed and Weinhold’s natural bond orbital (NBO) analysis18 implemented in Gaussian 03. 3. Results Small Models. Irrespective of the metal ion, the ground state of ES is characterized by one hydrogen bond between the metalbound hydroxide and the amide of the substrate and another hydrogen bond between the carbonyl oxygen of the formyl group and the amide of Leu91 (Figure 1, ES-gs) There is no ligation between the carbonyl oxygen of the formyl group and the metal center. Meanwhile, we also located another minimum of the ES complex (ES-m2) for Ni2+ and Fe2+ in which the carbonyl oxygen binds the metal center without the hydrogen bond between the metal-bound hydroxide and the amide of the substrate. These minima are about 8.8 kcal/mol higher in energy than the corresponding ground states (Figure 1, ES-gs). On the other hand, repeated efforts to locate the similar metalcoordinated minimum for Zn2+ failed, indicating a weaker interaction between the carbonyl oxygen and Zn2+ compared to Ni2+ and Fe2+. These results are consistent with Goldberg’s observations, in which Fe2+ prefers higher coordination than does Zn2+.8b As shown in Figure 1, the transition state was located for the initial nucleophilic attack of the metal-coordinated hydroxide on the carbonyl carbon. However, the conformations of the transition states are quite different for the three metal models.

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Figure 1. Optimized geometries of the complex and transition state of the small model; data depicted for Zn2+ (black), Ni2+ (blue), and Fe2+ (red). The relative energies are in kcal/mol for the ground state, minimum 2, and transition state for Zn2+, Ni2+, and Fe2+. The coordinated center is the metal ion.

Ni2+ and Fe2+ have a transition from a tetra- to a pentacoordinated metal center, whereas Zn2+ prefers tetracoordination rather than pentacoordination, as indicated by the distance between the metal center and the carbonyl oxygen Oδ (2.937 Å). Clearly, Zn2+ has little stabilization for the negatively charged Oδ. The calculated activation energies are 20.1, 14.3, and 16.8 kcal/mol for Zn2+, Ni2+, and Fe2+, respectively. Zn2+ has the lowest activity, which is qualitatively consistent with the experimental observations. Our model calculations indicate that the nucleophilic substitution step of the deformylation reaction is clearly dependent on the metal center, which is in contrast to the previous DFT calculations.8c As shown in Chart 1, formamide was used as the substrate in the previous calculations. The ground state was characterized by the carbonyl oxygen of formamide binding with the metal center, while the amide hydrogen at the syn position of the carbonyl group simultaneously formed a hydrogen bond with the metal-bound hydroxide. However, the syn position of the carbonyl oxygen of the formyl group should be a peptide backbone in the real system. The ground states in the previous calculations are thus unlikely to exist in the real system because of the steric constraints. Moreover, as described by our small models, Zn2+ has a weaker interaction with the carbonyl oxygen of the formyl group than Ni2+ and Fe2+. The metal-coordinated state of Zn model therefore has a higher energy than those of the Ni and Fe models. This higher energy decreases the activation energy of the Zn model in the previous calculation and attenuates the differences in the activities of the Zn, Ni, and Fe models. QM/MM Models. Although the small model that is truncated from the defomylase active site provides meaningful information about the catalytic reaction and the metal-ion dependence of the reactivity, it does not consider the protein environment and the real substrate. To fully characterize the reaction pathway and understand the metal-ion dependence of the reactivity, we further studied this reaction employing two-layered ONIOM models (PBE1PBE, Amber). As shown in Scheme 1, the deformylation reaction pathway includes several steps according to the mechanism of thermolysin and matrix metalloproteinases proposed by Siegbahn et al.:19 formation of an enymzesubstrate complex, nucleophilic addition of the metal-coordinated water/hydroxide to the carbonyl carbon of the formyl group, proton transfer, C-N bond breaking, and final release of the product. The optimized structures of the active species

and the reaction path are shown in Figures 3 and 4, and the calculated free energy profiles of the reactions are given in Figure 5 (see below). Enzyme-Substrate Complex. As shown in Figure 2, there are two distinct binding modes of the substrate in the active sites of PDFs. One binding mode is the tetracoordinated state (mode A) characterized by the carbonyl oxygen of the formyl group forming a hydrogen bond with the amide of Leu91 instead of coordinating with the metal. The terminal amide forms another hydrogen bond with Glu133. The metal-coordinated water forms strong hydrogen bonds with Glu133 and Gln50. In binding mode B, the carbonyl oxygen of the substrate binds strongly with the metal center but forms a relatively weaker hydrogen bond with the amide of Leu91. The terminal amide forms another hydrogen bond with the Gly45 backbone carbonyl oxygen rather than with Glu133 as in mode A. The metalcoordinated water shifts one proton to Glu133 and forms a metal-bound hydroxide. The relative energies of these two states for the Zn2+, Ni2+ and Fe2+ PDFs are given in Figure 2. Mode A is about 5 kcal/mol more favorable than mode B for ZnPDF, but mode B becomes more stable than mode A by about 0.3 and 1.2 kcal/mol for Fe-PDF and Ni-PDF, respectively. Thus, Zn-PDF prefers the tetracoordinated state as its ground state, whereas Fe-PDF and Ni-PDF prefer the pentacoordinated state as their ground states. These results indicate that Zn2+ has a stronger preference for tetracoordination than for pentacoordination, and higher coordination is more favored for Ni2+ and Fe2+, which is consistent with Chan et al.’s observations8a and Goldberg et al.’s results8b. It should be expected that the CdO bond of the formyl group is more polarized in Ni-PDF and Fe-PDF than in Zn-PDF in the ES complex, given that the carbonyl oxygen does not coordinate with Zn2+ in the ground state. This is reflected by the increase of the bond length from 1.232 Å in Zn-PDF to 1.244 Å in Ni-PDF and 1.241 Å in Fe-PDF. Moreover, the metalcoordinated water forms a strong hydrogen bond with Glu133 in Zn-PDF but is not deprotonated (Figure 3a), whereas the metal-coordinated water is deprotonated by Glu133 in Ni- and Fe-PDFs (Figure 4a). Because the metal-coordinated water/ hydroxide acts as a nucleophile to attack the carbonyl carbon of the substrate formyl group, leading to the first transition state of the reaction, the deprotonation in Ni-PDF and Fe-PDF plays a crucial role in the deformylation activity, also demonstrating

Catalytic Mechanism of Peptide Deformylase

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SCHEME 1: Proposed Catalytic Cycle

the critical role of Glu133 in the catalytic process.20 The process described above also demonstrates the important role of the second-shell amino acids, such as Gly45, Gln50, and Leu91, in the binding modes of the substrate in the active site.6a,19 Transition State of Water/Hydroxide Nucleophilic Attack. The metal-coordinated water/hydroxide is well positioned for nucleophilic attack on the carbonyl carbon of the formyl group in peptide deformylases (Figures 3a, 4a). The distance between the oxygen, Oω, and carbon, Cδ, is 2.90 Å in Zn-PDF, 2.52 Å in Ni-PDF, and 2.53 Å in Fe-PDF. The nucleophilic attack proceeds via the transition state TSO-C (Figures 3b, 4b) to result in the tetrahedral intermediate INT1 (Figures 3c, 4c). The TSO-C geometries were obtained by a refined search around the maximum-energy point on the scanning potential energy surface (PES). They were confirmed to be transition states by having only one negative Hessian element, which corresponds to the normal mode of Oω-Cδ bond formation. The bond length of Oω-Cδ is about 1.63 Å for Zn-PDF and 1.75 Å for NiPDF and Fe-PDF, indicating that the transition state of ZnPDF is later than those of Ni- and Fe-PDFs. The corresponding calculated reaction barrier is 23.0 kcal/mol (Zn-PDF), 15.0 kcal/mol (Ni-PDF), and 14.9 kcal/mol (Fe-PDF). As shown in Figure 5, along the full reaction pathway, this step is the rate-determining step. Thus, the ONIOM results are in good agreement with the experimental observations, indicating that the Zn-PDF is roughly inactive, and the activation barrier is about 13.0-14.5 kcal/mol for the Ni2+ and Fe2+ forms of PDF,

as estimated from the experimental kcat values (210-2100 s-1)6b,6d using transition state theory.21 Moreover, a proton shifts from the Zn-coordinated water to Glu133 in concert with OωCδ bond formation in Zn-PDF. The newly developed negatively charged oxygen, Oδ, is stabilized by the amide of Leu91 and the metal center accompanied by a transition from a tetra- to a pentacoordinated metal center. Tetrahedral Intermediate. The TSO-C transition state leads to a stable intermediate, INT1 (Figures 3c, 4c). The structures of the intermediate INT1 are similar for Ni-PDF and Fe-PDF, in which the Oω-Cδ bond becomes shorter than that in TSO-C, but the Cδ-Nδ bond becomes longer as a result of the transition from sp2 hybridization to sp3 for Cδ. At the same time, the hydrogen bond between Glu133 and Oω of the hydroxyl group partially switches from Oω to Nδ because of the increase of the basicity of Nδ (Figure 4c). However, the intermediate INT1 of Zn-PDF again exhibits obvious differences from those of NiPDF and Fe-PDF, in which the proton of the Glu133 is completely shifted to Nδ (1.01 Å) and the Oω-Cδ bond becomes even shorter (1.394 Å) than those in Ni-PDF and Fe-PDF (1.519-1.595 Å), whereas the Cδ-Nδ bond is longer (1.597 Å) than those of Ni-PDF and Fe-PDF (1.409-1.459 Å). Another structural feature is that the proton of the Zncoordianted hydroxyl group transfers to Glu133. Along the full reaction pathway, INT1 of Zn-PDF resembles INT2 of NiPDF and Fe-PDF (Figure 4e). However, the proton-transfer processes of Ni-PDF and Fe-PDF have to occur through a

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Figure 2. Structural view of the active site, with the important elements and interaction of catalysis. Left is mode A (tetracoordinated state), in which the carbonyl oxygen of the formyl group does not bind with the metal center; right is mode B (pentacoordinated state), in which the carbonyl oxygen of the formyl group binds with the metal center. The relative energies (kcal/mol) for the two modes are presented at the bottom. The lengths of the hydrogen bonds between the carbonyl oxygen of the substrate and the amide of Leu91 for Zn-PDF, Ni-PDF, and Fe-PDF in mode A are 1.907, 1.894, and 1.890, respectively.

Figure 3. Optimized geometries of QM regions of species on the energy profile of Zn-PDF.

transition state, TSH-N, in which the proton is between the oxygen of Glu133 and the Nδ atom with partial linking to both atoms (Figure 4d), whereas the calculated corresponding activation energies are only about 0.9 and 3.5 kcal/mol for Ni-PDF and Fe-PDF, respectively. Indicating that these are fast processes. Breaking of the Cδ-Nδ Bond and Formation of the Final Products. Proton transfer makes the -NHR group a good leaving group. Meanwhile, the length of the Oω-Cδ bond is reduced from 1.40 to 1.32 Å, with partial double-bond character.

The proton of the metal-coordinated hydroxyl group transfers to the carboxyl group of Glu133. In the final product, EP, the Cδ-Nδ bond is completely broken, and the average bond length changes from 2.0 Å in TSC-N to about 3.0 Å in EP. The proton of the N-terminal amine group of the released product forms a strong hydrogen bond with Glu133 (1.80-2.1 Å for the HωO distance). The produced formate group binds to the metal center and also forms a hydrogen bond with the amide of Leu91. The proton of the formate group transfers to Glu133 automatically as the Cδ-Nδ bond breaks. Intriguingly, the binding mode

Catalytic Mechanism of Peptide Deformylase

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Figure 4. Optimized geometries of QM regions of species on the energy profiles of (top) Ni-PDF and (bottom) Fe-PDF.

Figure 5. Energy profiles for deformylation catalyzed by Zn-, Ni-, and Fe-PDF.

of the formate group with the metal center for Zn-PDF is significantly different from that for Ni-PDF and Fe-PDF. In Zn-PDF, the formate binds to Zn2+ in a monodentate fashion, whereas Oω of the formate group coordinates with the metal center at a distance of 2.005 Å, but another oxygen has a distance of 2.839 Å from the metal center (Figure 3d). In contrast, in Ni-PDF and Fe-PDF, formate coordinates with the metal center in a bidentate fashion, with the two O-M distances being about 2.123 and 2.149 Å for Ni-PDF and about 2.294 and 2.147 Å for Fe-PDF. Such results are in excellent agreement with the recent high-resolution X-ray crystal structure of E. coli PDF complexed with formate8a (Table S1), in which

the M2+-O distances are about 2.09 and 2.88 Å for Zn-PDF and 2.44 and 2.30 ( 0.17 Å for Fe-PDF. Thus, the two-layered ONIOM method used in this work can reasonably address the structural and energetic features of this enzymatic system. 4. Discussion As described above, the results of the ONIOM study presented in this work provide a detailed and insightful description of the reaction pathway of the deformylation process catalyzed by peptide deformylases. They also account for the metal dependence of the activity of PDF.

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TABLE 1: Molecular Orbital (MO) Energies (eV) of Activated Fragments of the Active Site for Zn-PDF, Ni-PDF, and Fe-PDF mode A

TABLE 2: Wiberg Bond Indexes (WBI) of the Metal-Ligand Bonds for Zn-, Ni-, and Fe-PDFs in Mode A, Mode B, and TSO-C and NBO Charges of the Metal Ions

mode B

Zn-PDF

PDF

HOω (HOMO)

π*CdOδ (LUMO)

HOω (HOMO)

π*CdOδ (LUMO)

Zn-PDF Ni-PDF Fe-PDF

-8.853 -9.228 -9.006

1.255 1.324 1.319

-7.295 -7.065 -7.490

0.566 0.463 0.221

Influence of the Preference for the Coordination Geometry on the Reactivity. Figure 2 depicts the ground states of Zn-PDF, Ni-PDF, and Fe-PDF, as well as the relative energies of the alternative binding mode of the enzymesubstrate complexes. Zn-PDF clearly prefers tetracoordinated metal centers (mode A), whereas pentacoordinated metal centers (mode B) are favorable for Ni-PDF and Fe-PDF. The two modes have significantly different influences on the reactivity of the active site. In mode A, the metal center acts as a Lewis acid to polarize the coordinated water, but the basicity of Glu133 is lessened by the hydrogen bond between the substrate and Glu133. The metal-coordinated water cannot be fully deprotonated to form metal-coordinated hydroxide. Meanwhile, the carbonyl of the formyl group cannot be activated by the metal without metal coordination. In mode B, the metal center acts as a Lewis acid to polarize both the nucleophile and the substrate. Without the hydrogen bond from the terminal amide of the substrate, Glu133 can completely deprotonate the metalcoordinated water to form a metal-coordinated hydroxide. On the other hand, the metal center also activates the carbonyl group of the formyl group of the substrate, which is reflected by the increased CdOδ bond distances of 1.236 Å (Zn2+), 1.244 Å (Ni2+), and 1.241 Å (Fe2+), compared to those of mode A of 1.232 Å (Zn2+), 1.232 Å (Ni2+), and 1.231 Å (Fe2+), respectively. Molecular orbital calculations of these models indicate that mode A has a lower molecular orbital energy for the p lone-pair electrons of the water oxygen Oω and a higher molecular orbital energy for the anti-π bond of the carbonyl group of substrate (Table 1, Figure S4) compared to mode B. According to frontier molecular orbital (FMO) theory,22 the smaller energy gap between the nucleophile HOMO and the carbonyl LUMO in mode B compared to mode A makes mode B the activated precursor state for the nucleophilic attacking step. Thus, Zn-PDF has to overcome about 5 kcal/mol in energy to go from the ground state (mode A) to the activated precursor state (mode B), whereas the ground states of Ni-PDF and FePDF are already in the activated precursor state for the successive reaction step. We can expect that Ni-PDF and FePDF have higher reactivities than Zn-PDF in the ground state. The results shown in Figure 5 also indicate an intrinsically lower reactivity of Zn compared to Ni and Fe. Specifically, the calculated activation energy for Zn-PDF is about 17.8 kcal/ mol with respect to the mode B reactant, compared to 15.0 and 14.9 kcal/mol for Ni-PDF and Fe-PDF, respectively. This is also qualitatively reflected by the calculated HOMO-LUMO gaps in mode B, as listed in Table 1. Zn-PDF has a larger gap than do Ni-PDF and Fe-PDF. It therefore has a lower reactivity for the nucleophilic addition of hydroxide to the carbonyl substrate. Origin of the Coordination Geometry Preference. The carbonyl oxygen of the substrate forms a strong hydrogen bond with the amide of Leu91 of the active site in mode A, whereas it forms a metal-oxygen bond in mode B. Thus, the preferences for mode A and mode B are determined by the relative strengths of these two types of binding. The hydrogen bonds in mode A

Ni-PDF

Fe-PDF

0.4103 0.1361 0.1442 0.1545 0.0035 0.8486 1.293

0.4534 0.1287 0.1373 0.2429 0.0033 0.9656 1.316

0.3613 0.1154 0.1104 0.1527 0.1014 0.8412 1.325

0.442 0.1209 0.1032 0.2383 0.1078 1.0122 1.345

0.3441 0.1108 0.1142 0.1029 0.1485 0.8205 1.290

0.4104 0.1165 0.1184 0.1373 0.1774 0.9600 1.344

mode A WBIM-S90 WBIM-N132 WBIM-N136 WBIM-Oω WBIM-Oδ WBItotal chargemetal

0.4012 0.0995 0.1084 0.1179 0.0023 0.7293 1.466

WBIM-S90 WBIM-N132 WBIM-N136 WBIM-Oω WBIM-Oδ WBItotal chargemetal

0.3418 0.0853 0.0830 0.1337 0.0503 0.6941 1.461

WBIM-S90 WBIM-N132 WBIM-N136 WBIM-Oω WBIM-Oδ WBItotal chargemetal

0.3100 0.0831 0.0947 0.0558 0.1124 0.6560 1.482

mode B

TS

of Zn-PDF, Ni-PDF, and Fe-PDF are roughly identical, reflected by similar bond distances and bond angles (Figure 2, at the bottom). Therefore, the different preferences for mode A and mode B in Zn-PDF, Ni-PDF, and Fe-PDF are obviously influenced by the strengths of the metal-oxygen bonds in their active sites. The chemical bonding in the transition metal complex is commonly described in terms of the ionic and covalent interactions between the metal and the ligands,23a and a detailed discussion of the correlation between the metalligand bond properties and the nuclear charge of metal center is provided by Gorelsky et al.23b Because of electrostatic neutralization by the negatively charged Cys90 and the deprotonated hydroxide, the positive charge of the metal center is reduced significantly from 2+. It is expected that the metal center would have a relatively weak electrostatic attraction with the incoming neutral carbonyl oxygen of the substrate whereas the strength of the metal-oxygen bond is highly influenced by the covalent interactions. The effective nuclear charge of the metal increases from Fe to Ni to Zn. Thus, the energy gap between the metal d-based MOs and the carbonyl oxygen p-based MOs increases from Fe to Zn (Figure S5). In tetracoordinated mode A, this gap is about 5.0 eV for Zn, but it is only about 2.0 eV for Ni and Fe; it is therefore expected that there will be stronger orbital interaction between the metal and the carbonyl oxygen for Ni and Fe than Zn in mode B. In addition, Zn has completely filled d orbitals (d10), but Ni and Fe have partially filled d orbitals that can interact with the oxygen lone pairs. As a result, there is only a weak interaction between Zn and the carbonyl group, as indicated by the small Wiberg bond index17 of 0.050 for the Zn-Oδ bond (Table 2). For Ni and Fe, the interaction with the carbonyl of the substrate is stronger, as shown by the increased Wiberg bond indices of 0.101 and 0.108 for the Ni-Oδ and Fe-Oδ bonds, respectively (Table 2). The weaker coordination interaction of the carbonyl group with Zn compared to Ni and Fe is also indicated by the fact that ES-m2 can be located only for Ni and Fe in the small model (Figure 1). Table 2 clearly shows that Zn has a higher ionic character and a lower covalent character than Ni and Fe.

Catalytic Mechanism of Peptide Deformylase The situation of ligand binding with the metal center in the transition state is similar to that of mode B. The Zn-Oδ bond has the smallest Wiberg bond index; the Fe-Oδ bond has the largest Wiberg bond index; and the Wiberg bond index of the Ni- Oδ bond is in between, as reported in Table 2. These results are closely correlated with the calculated activation energy of the nucleophilic attack step, in which Zn2+ has a much higher activation energy (∼23.0 kcal/mol) in the rate-determining step than Ni2+ (15.0 kcal/mol) and Fe2+ (14.9 kcal/mol), which is in good agreement with the experimental observations for the E. coli PDFs, in which Zn-PDF has no observable catalytic activity and Ni-PDF and Fe-PDF have comparable catalytic activities.6d These results led us to the conclusion that the ligand environments and the intrinsic electronic structures of the metal centers in the active sites of PDFs determine the coordination geometry preference and hence the catalytic activity. Comment on the Activity of AtPDF1A and LiPDF. Although this model can be satisfactorily applied to E. coli PDFs, it is partially disputed by the recently discovered Arabidopsis thaliana PDF1A (AtPDF1A)24 and Leptospira interrogans PDF (LiPDF),25 whose Zn forms are almost as efficient as Ni-PDF and Fe-PDF from E. coli. Comparison of the overall structures of AtPDF1A24b and LiPDF25c with E. coli PDFs reveals a conservation of the PDF folds, with the exception of the C-terminal region. The active site is also well conserved, especially in terms of metal chelation, which implies that the catalytic mechanisms of AtPDF1A, LiPDF, and E. coli PDFs should be identical. The substrate binding sites of AtPDF1A and LiPDF are significantly different from that of E. coli PDFs. The former have an extra S3′ binding pocket that possibly facilitates the binding of the substrate in AtPDF1A. The flexible CD loop of LiPDF was observed to have open and closed conformations suggested to facilitate the substrate binding.25c Thus, enhancement of the catalytic efficiency for AtPDF1A and LiPDF might result from the stronger binding of the substrate to the enzyme. This is reflected by a comparison of the kcat, Km, and kcat/Km values.24a,25b The kcat value of AtPDF1A is higher than that of Zn-PDF (E. coli), but still about 10 times lower than that of Ni-PDF (E. coli). However, the Km value of AtPDF1A is about 10-fold lower than that of E. coli Ni-PDF and even about 300-fold lower than that of E. coli Zn-PDF. The kcat value of LiPDF is similar to that of ZnPDF (E. coli), but the Km value of LiPDF is about 30 times lower than that of E. coli Zn-PDF. Therefore, the catalytic efficiencies of AtPDF1A and LiPDF are comparable to that of E. coli Ni-PDF but much higher than that of E. coli Zn-PDF. Thus, our calculated mechanism and the understanding of the metal dependence of the activity of the E. coli PDFs also apply to AtPDF1A and LiPDF, but we need to take into account the influence of some subtle structural differences between E. coli PDFs, LiPDF, and AtPDF1A in enzyme activities. Another question concerning the activity of Zn-PDF also needs to be considered: Why is the activity of Zn-PDF much lower than those of other zinc metalloenzymes such as thermolysin, even though they share similar active sites? The structural comparison of the active sites of Zn-PDF and thermolysin, along with a previous theoretical study on thermolysin,19a can provide some insight into understanding this question. In addition to the zinc ion, thermolysin has a His231 residue around the active site to activate the substrate and stabilize the intermediate. Mutation of the His231 of thermolysin to Ala would cause about 1000-fold decrease in activity,26 and an increase of more than 10 kcal/mol in activation barrier was reported based on computer modeling.19a Such residue assistance

J. Phys. Chem. B, Vol. 111, No. 22, 2007 6243 in activity is also highlighted in other active zinc metalloenzymes.4,27 However, Zn-PDF does not have such a residue or alternative, which can partially account for the low activity of Zn-PDF. 5. Conclusions The complete pathway of the PDF-catalyzed deformylation reaction has been described by a two-layerd ONIOM (PBE1PBE, Amber) study. The reaction cycle includes several steps: formation of an enymze-substrate complex, nucleophilic addition of the metal-coordinated water/hydroxide, proton transfer, C-N bond breakage, and final release of the product. Our results indicate that the nucleophilic addition of the metalcoordinated water/hydroxide to the carbonyl carbon of the formyl group resulting in a tetrahedral intermediate is the ratedeterminng step in the deformylation. Zn-PDF has a much higher activation energy (∼23.0 kcal/mol) than Ni-PDF (15.0 kcal/mol) and Fe-PDF (14.9 kcal/mol). These results are in good agreement with experimental observations. The lower catalytic activity of Zn-PDF is mainly attributed to two factors: the unfavorable pentacoordination (mode B), as suggested earlier,8a-b contributes about 5-6 kcal/mol higher activation energy, and the lower intrinsic reactivity of Zn than Ni and Fe contributes another 2-3 kcal/mol higher activation energy. Acknowledgment. We are grateful to the National Science Foundation of China (20225312) for financial support of the research. Supporting Information Available: Additional details on the scanning potential surfaces of transition states for Zn-, Ni-, and Fe-PDFs; geometrical comparison of the active sites of ONIOM calculations; and X-ray crystal structures. Bond lengths and angles of two hydrogen bonds in model A. Contour surfaces and orbital energies of frontier molecular orbitals of the active sites. This material is available free of charge via the Internet at http://pubs.acs.org References and Notes (1) (a) Meinnel, T.; Mechulam, Y.; Blanquet, S. Biochimie 1993, 75, 1061. (b) Giglione, C.; Meinnel, T. Trends Plant Sci. 2001, 6, 566. (c) Bradshaw, R. A.; Brickey, W. W.; Walker, K. W. Trends Biochem. Sci. 1998, 23, 263. (2) (a) Mazel, D.; Pochet, S.; Marliere, P. EMBO J. 1994, 13, 914. (b) Giglione, C.; Pierre, M.; Meinnel, T. Mol. Microbiol. 2000, 36, 1197. (3) (a) Chen, P. S.; Patel, D. V.; Hackbarth, C. J.; Wang, W.; Dreyer, G.; Young, D. C.; Margolis, P. S.; Wu, C.; Ni, Z.; Trias, J.; White, R. J.; Yuan, Z. Biochemistry 2000, 39, 1256. (b) Yuan, Z.; Trias, J.; White, R. J. DDT 2001, 6, 954. (c) Apfel, C.; Banner, D. W.; Bur, D.; Dietz, M.; Hirata, T.; Hubschwerlen, C.; Locher, H.; Page, M. G. P.; Prison, W.; Rosse, G.; Specklin, J. J. Med. Chem. 2000, 43, 2324. (d) Howard, M. H.; Cenizal, T.; Gutteridge, S.; Hanna, W. S.; Tao, Y.; Totrov, M.; Wittenbach, V. A.; Zheng, Y. J. Med. Chem. 2004, 47, 6669. (e) East, S. P.; Beckett, R. P.; Brookings, D. C.; Clements, J. M.; Doel, S.; Keavey, K.; Pain, G.; Smith, H. K.; Thomas, W.; Thompson, A. J.; Todd, R. S.; Whittaker, M. Bioorg. Med. Chem. Lett. 2004, 14, 59. (f) Molteni, V.; He, X.; Nabakka, J.; Gordon, P.; Bursulaya, B.; Warner, I.; Shin, T.; Biorac, T.; Ryder, W. S.; Goldberg, R.; Doughty, J.; He, Y. Bioorg. Med. Chem. Lett. 2004, 14, 1477. (g) Cali, P.; Nærum, L.; Mukhija, S.; Hielmencrantz, A. Bioorg. Med. Chem. Lett. 2004, 14, 5997. (4) Lipscomb, W. N.; Strater, N. Chem. ReV. 1996, 96, 2375. (5) (a) Vallee, B. L.; Auld, D. S. Biochemistry 1990, 29, 5647. (b) Meinnel, T.; Blanquet, S. J. Bacteriol. 1993, 175, 7737. (c) Meinnel, T.; Blanquet, S. J. Bacteriol. 1995, 177, 1883. (6) (a) Chan, M. K.; Gong, W.; Rajagopalan, P. T. R.; Hao, B.; Tsai, C. M.; Pei, D. Biochemistry 1997, 36, 13904. (b) Rajagopalan, P. T. R.; Yu, X. C.; Pei D. J. Am. Chem. Soc. 1997, 119, 12418. (c) Ragusa, S.; Blanquet, S.; Meinnel, T. J. Mol. Biol. 1998, 280, 515. (d) Becker, A.; Schlichting, I.; Kabsch, W.; Schultz, S.; Wagner, A. F. V. J. Biol. Chem. 1998, 273, 11413. (e) Groche, D.; Becker, A.; Schlichting, I.; Kabsch, W.;

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