Heme Electron Transfer in Peroxidases: The Propionate e-Pathway

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J. Phys. Chem. B 2008, 112, 13460–13464

Heme Electron Transfer in Peroxidases: The Propionate e-Pathway Victor Guallar* ICREA Research Professor, Life Science Department, Barcelona Supercomputing Center, Jordi Girona, 29, 08034 Barcelona (Spain) ReceiVed: July 21, 2008

Computational modeling offers a new insight about the electron transfer pathway in heme peroxidases. Available crystal structures have revealed an intriguing arrangement of the heme propionate side chains in heme-heme and heme-substrate complexes. By means of mixed quantum mechanical/molecular mechanics calculations, we study the involvement of these propionate groups into the substrate oxidation in ascorbate peroxidase and into the heme to heme electron transfer in bacterial cytochrome c peroxidase. By selectively turning on/off different quantum regions, we obtain the electron transfer pathway which directly involves the porphyrin ring and the heme propionates. Furthermore, in ascorbate peroxidase the presence of the substrate appears to be crucial for the activation of the electron transfer channel. The results might represent a general motif for electron transfer from/to the heme group and change our view for the propionate side chains as simple electrostatic binding anchors. We name the new mechanism “the propionate e-pathway”. Introduction Heme proteins are ubiquitous across the biological world and play a key role in electron transfer and metabolic oxidation.1,2 The electronic structure and the electron transfer pathway of their highly reactive intermediates are the focus of substantial research.3-7 Of particular interest is compound I (Cpd I)a, an oxyferryl species known to be one of the major players in peroxidase oxidative catalysis. Cpd I is an FeIV species with two unpaired electrons in an iron-oxo moiety and a third unpaired electron in a porphyrin/protein radical.8,9 The final steps of Cpd I formation, in both hydrogen peroxide and molecular oxygen activations, are believed to follow protonation of the distal oxygen and release of a water molecule.10,11 This process is schematically shown in Figure 1. The O-O bond breaking, driven by the distal oxygen protonation, creates a formal FeV species, an extremely electron deficient metal center, capable of forming protein/porphyrin radicals and substrate oxidation. The electron transfer pathway into the heme center and the nature of the third unpaired electron has been the center of intense discussions.4,12 The right frame in Figure 1 indicates the substrate (ascorbate) oxidation in the ascorbate peroxidase (APX) enzyme. It also depicts the substrate arrangement in the crystal complex: in direct contact with the heme propionates, substantially removed from the iron center.13 Additional evidence of the importance of the propionates in APX comes from mutation studies. Barrows et al. observed a migration of the third unpaired electron from the porphyrin to a Trp radical when modifying the propionate screening.14 This radical delocalization has also been proposed by Raven et al. when observing different covalent linkages to the heme group.15,16 Barrows studies were motivated by recent work showing spin delocalization into the heme propionates in cytochrome P450.17,18 The delocalization mechanism involves a delicate balance between the metal d * To whom correspondence should be addressed. E-mail: [email protected]. aAbbreviations: QM/MM: quantum mechanics/molecular mechanics, APX: Ascorbate peroxidase, Cpd I: compound I, CcP: Cytochrome C peroxidase, DFT: Density functional theory, bCcP: Bacterial Cytochrome C peroxidase, mKatG: bifunctional catalase-peroxidase

Figure 1. Cpd I formation. Final steps in Cpd I formation involving Compound 0 (Cpd 0) and the protonated Compound 0. The right frame indicates the arrangement of the ascorbate and the heme 6-propionate. His refers to the axial histidine connecting the heme to the protein.

orbitals, the porphyrin orbitals, and the propionate’s oxygens lone pairs. This balance is highly dependent on the electrostatic environment of the heme. In P450cam, for example, we showed that the nature of the third unpaired electron in Cpd I drastically changed upon different protonation states of Asp29712 (results reproduced later independently 19). Further evidence of the delicate balance affecting the nature of the third unpaired electron comes from several apoenzyme experiments showing different spin distributions on closely related active sites. Cytochrome c peroxidase (CcP), ascorbate peroxidase (APX), and the bifunctional catalase-peroxidase (mKatG) enzymes constitute a good example of this diversity. They have very similar active sites, but the third unpaired electron resides in the proximal tryptophan residue in CcP (Trp191), in the porphyrin ring in APX, and in the distal tryptophan in mKatG (Trp107).15 Remarkable crystallographic support for the active role of the propionates comes from the bacterial diheme cytochrome C peroxidase (bCcP) from Pseudomonas Nautica 617. bCcP is one of the simplest prototypes for multicenter electron transfer proteins, containing a high potential electron-transferring heme (left heme in Figure 2) and a low potential peroxidatic heme (right heme in Figure 2). The catalytic peroxidatic heme follows a hydrogen peroxide decomposition biochemistry. The enzyme activation event, observed crystallographically by Dias et al., is triggered by an essential calcium ion.20 Upon activation, there is a large rearrangement resulting in a direct link connecting the two heme centers through the propionates and Trp94. The

10.1021/jp806435d CCC: $40.75  2008 American Chemical Society Published on Web 09/25/2008

The Propionate e-Pathway

Figure 2. Inactive (green) and active (orange) X-ray structures from Pseudomonas Nautica 617 cytochrome C peroxidase. The bridging tryptophane and the high- and low-potential heme groups are labeled.

cores for the active and inactive crystallographic structures are shown in Figure 2. Here, we present a direct evidence of the propionate lone pairs active role in Cpd I electron transfer pathway for APX and bCcP. We employ mixed quantum mechanics/molecular mechanics (QM/MM) methods to describe the electronic structure of the intermediates shown in Figure 1.21 To track the electron transfer pathway, we use a novel approach based on adding/deleting components to the quantum region. In QM/MM methods, the Schro¨dinger equation is only solved for those atoms included in the quantum region (QM). The MM or classical region is described by a set of solid spheres with point charges. Thus, electrons can only flow between those atoms in the QM region. The results indicate an electron pathway connecting the electron donor and acceptor through the propionate lone pairs, supporting the direct path observed in the crystallographic structures. We have also performed calculations on the apoenzymes proposed above, APX, CcP, and mKatG, to check for the accuracy of the QM/MM spin densities. For each system, we obtain a spin density in good agreement with the experimental observations, showing that QM/MM methods are an excellent tool for the analysis of the radical heme species. Computational methods QM/MM methods can join together QM and MM representations of different sectors of a complex system.21,22 The conjunction of these technologies contains the elements necessary to properly describe the potential energy surfaces relevant to enzymatic chemistry. The models for the compound I apoenzymes, APX, CcP, and mKatG where extracted from the 1OAG, 2CYP, and 1SJ2 pdb structures, respectively. The closest crystallographic water molecule to the iron center was used to model the oxo ligand. The substrate bound APX and the diheme bCcP models were obtained from the 1OAF and the 1RZ5 crystals, respectively. Water 501 and 563 were used to model the H2O2 group. According to its pKa, ascorbate was modeled with one negative charge, forming a hydrogen bond with the heme 6-propionate. The crystallographic O1D(propionate)O2(ascorbate) distance is 2.58 Å, indicating the existence of a hydrogen bond between them. In addition, the crystallographic NH2(Arg172)-O3(ascorbate) distance is 2.87 Å. Thus, the protonation state of the ascorbate substrate is clearly assigned from the crystal structure. Preparation of the system included soaking the protein in a box of pre-equilibrated water molecules followed by 100 ps molecular dynamics at 300 °K using periodic boundary conditions. The system was slowly heated and cooled in the first and last 10 ps. (More general details on the methods and system preparation has been published elsewhere.21) For the QM/MM

J. Phys. Chem. B, Vol. 112, No. 42, 2008 13461 minimizations, only a 10 Å layer of water molecules surrounding the proteins was kept. Geometry optimizations were carried out using unrestricted DFT with the B3LYP functional in combination with the 6-31G* (LACVP* effective core potential for the iron) basis set. In all optimizations, everything is free to move but the last layer of oxygens from the explicit water solvent, which was constrained. For all intermediates, we calculated the quartet and doublet spin states. All calculations were performed with the Qsite program.23 For the modeling of the apo-enzymes APX, CcP, and mKatG, the quantum region includes the heme group, the axial histidine (covalent link of the heme group to the protein), the oxo ligand, and the distal and axial tryptophan residues. For the APX system with bound ascorbate, we modeled two different quantum regions. The large region includes the heme group, the axial histidine, the H2O2 axial ligand, the proximal histidine, and the ascorbate substrate. The smaller quantum region excludes the ascorbate substrate from the larger one. For the diheme bCcP system, we have modeled three different quantum regions. The larger quantum region includes both heme groups, all covalently attached residues to each heme, the H2O2 axial ligand, and the bridging tryptophan, Trp 94. The medium quantum region excludes from the large one the high potential-electron transferring heme and all residues covalently attached to this heme group. The smaller quantum region excludes from the medium one the bridging Trp 94. The results presented below are based on spin density plots describing the radical content of the different intermediates. The spin plots are obtained from a grid projection of the difference in the alpha and beta spin-orbitals from an unrestricted wave function. They represent the total amount (and localization) of spin density. A Mulliken population analysis has also been used to approximately quantify the amount of radical character in specific groups, like the metal center, the ascorbate substrate, for example. Results Modeling of Cpd I spin density for the CcP, APX, and mKatG enzymes is shown in Figure 3. The mulliken and the spin-density analysis reveals, as expected, two unpaired electrons located in the d and p orbitals of the iron-oxo moiety; the main difference is the nature of the third unpaired electron. For Cpd I, in agreement with previous theoretical results, both quartet and doublet spin states are degenerate with a very similar spindensity population (besides the spin sign, antiferromagnetically coupling for the doublet spin state).24 For the CcP system, the third unpaired electron is localized at the proximal Trp. In mKatG, the electron is mostly localized in the porphyrine group but with a notable contribution in the distal tryptophan. In the APX enzyme, the unpaired electron is more delocalized, with a main contribution in the porphyrine ring but with a small component in the proximal Trp and a minimal one in the distal Trp. As discussed below, the theoretical modeling obtained with QM/MM methods largely agrees with the experimental knowledge of the spin distribution for the three systems. Figure 4 shows Cpd 0 and Cpd I spin densities for the larger QM region in the substrate bound APX system, including the ascorbate substrate, the heme group, the H2O2 ligand, and the proximal and axial histidines, His 163 and His 42. In this study, we are not focusing on obtaining the relative energies between the different intermediates (and transition states) but the electronic structure and the possible electron delocalization. Our results, however, confirmed previous studies where Cpd 0 was found to be a doublet state with the lowest quartet state ∼10

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Guallar

Figure 3. Spin densities for the Cpd I quartet spin state for the APX (left panel), mKatG (middle panel), and CcP (right panel) apo-enzymes. The proximal and distant tryptophans are labeled for clarity.

Figure 4. Spin density for the doublet Cpd 0 and Cpd I using the large quantum region including the ascorbate substrate. Positive and negative spin densities are depicted in blue and red, respectively. The porphyrin and the substrate are shown in a stick representation and atomtype coloring. The OOHH ligand lies above the porphyrin plane.

kcal/mol higher in energy.25,26 This energy gap is reduced along the O-O dissociation, being both states almost degenerate for Cpd I, where the doublet spin state presents three unpaired electrons (antiferromagnetically coupling as seen with the different colors in Figure 4). Cpd 0 has most of the unpaired electron density located at the iron center, left panel in Figure 4. The plot of the spin density nicely depicts that of a dxz orbital (or dyz, if we take the z axis as the axial ligand). No significant traces of spin density are seen beyond the first coordination shell of the metal center. The spin delocalization, however, changes substantially in the active species. The mulliken population analysis and the plot of the spin density for Cpd I reveal two unpaired electrons in the iron-oxo moiety and one more unpaired electron in the ascorbate substrate, right panel in Figure 4. The spin density on the metal center (and oxo atom), consistent with previous studies on Cpd I models 27-29 shows a nice dxz-dyz orbital mix (px-py mix for the oxo atom). The additional unpaired electron (the third unpaired electron), mainly found at the porphyrin π system in reduced model compounds,25,27,28,30 is fully localized at the π system of the ascorbate substrate; as expected a full electron has been transferred from the substrate to the metal center. The electron transfer is accompanied with the proton transfer from the ascorbate to the 6-propionate, for a total net effect of a hydrogen atom transfer. To analyze the electron-transfer pathway from the substrate to the metal center, associated to a clear change in the radical nature of the active site, we have studied the spin delocalization along the ligand O-O bond breaking. As seen by Wirstam et al., when the O-O bond distance is elongated the spin density increases on both oxygens.25 At short O-O distances, however, there is no indication of spin density in the porphyrin or in the substrate. At a O-O distance of ∼1.9 Å, the first spin delocalization appears: a significant component of spin density on the substrate. The right panel in Figure 5 shows the spin density for this intermediate structure, where we see a nascent

Figure 5. Spin density for a doublet spin state intermediate along Cpd I formation for two different quantum regions. Positive and negative spin densities are depicted in blue and red, respectively. The OOHH ligand lies above the porphyrin plane.

radical component in the π system of the ascorbate substrate. For the iron-oxo moiety, the spin density now depicts a mix of dxz, dyz, and oxygen p orbitals; we see no traces of spin density on the porphyrin ring. By employing mixed QM/MM methods, we can switch on and off the electronic description of different regions in the enzyme (the quantum region). The left panel of Figure 5 displays the spin density for the same intermediate (same structure as the right panel) but with the ascorbate substrate excluded from the quantum region. Eliminating the quantum description of the ascorbate eliminates the possibility of an electron transfer from this species to the iron metal center. As seen in Figure 5, at this point along the O-O elongation reaction coordinate and using this reduced quantum region we also see spin density beyond the metal coordination. The additional spin delocalization is now centered on the porphyrine ring with a remarkable component in the propionate group next to the ascorbate substrate. The black arrow in the left panel in Figure 5 points to this significant component of radical character in the lonepair orbitals for the heme propionate side chains. We should mention here that analogous results for the Cpd I spin density are obtained when studying the quartet spin state (but with only one sign spin density). We have also studied the effects of changing the protonation state of His169, the only controversial protonation center upon close inspection of the crystal structures. The protonation state of this residue is not clear because it is located next to two arginines but also next to the propionates and to the ascorbate substrate. Both studies, with a neutral and a protonated histidine, gave the same qualitative spin densities. The only difference is that for the protonated histidine the electron transfer from the substrate appears earlier along the O-O coordinate. The dependency of the spin density with protein fluctuations, an important aspect largely emphasized in previous studies,31,32 has been studied. We performed single-point QM/MM calculations, from 10 different snapshots along a 1 ns molecular dynamics trajectory. Figure 1 in the Supporting Information shows the first, middle, and last snapshot spin densities. We can observe that most spin density for the third unpaired electron

The Propionate e-Pathway

J. Phys. Chem. B, Vol. 112, No. 42, 2008 13463 the quantum region, we see an important contribution of the Trp94 residue to the spin density. For the medium and small quantum regions, the lone pairs of the propionate in the catalytic heme present radical character, highlighted with two black arrows in Figure 6. Recent studies by Brittain et al. have also observed the presence of spin density in the Trp94 and in the high potential heme. These studies, however, were conducted in the gas phase and the propionates were neutralized.33 We have also performed calculations with more approximate electron transfer pathway software. We have used Harlem34 and Greenpath35 to map the electron pathway and obtained very similar results. Figure 2 in the Supporting Information shows the pathways obtained by using Harlem in APX and bCcP. Discussion

Figure 6. Cpd I spin density for three different quantum regions in bCcP. The spin density corresponds to the quartet spin state. The high potential heme (HP) and the low potential one (LP) are labeled for clarity.

is localized in the ascorbate substrate, in agreement with the full minimized crystal structure. We have performed a similar analysis for the diheme cytochrome C peroxidase from Pseudomonas Nautica 617. The results along the hydrogen peroxide O-O elongation coordinate indicate comparable results to those of APX. The O-O bond breakage results in a highly deficient iron center and activates the electron transfer between the heme metal centers. To illustrate the electron transfer pathway between the two heme centers, we show the spin delocalization for compound I obtained at three different quantum regions. The top panel in Figure 6 illustrates the spin density for the large quantum region: including both hemes, all protein residues covalently attached to the hemes, the H2O2 ligand (at this stage transformed into the oxo ligand plus the leaving water molecule), and the bridging Trp94. To demonstrate the equivalence between the quartet and doublet spin densities, the quartet spin density results are shown this time. The spin density analysis displays two unpaired electrons located exclusively at the iron-oxo moiety in the low potential catalytic heme (right heme, LP, in Figure 6), with the third unpaired electron residing in the other heme group (high potential heme, HP, in Figure 6). As in the APX system, the two unpaired electrons in the catalytic heme are located in a dxz-dyz orbital mix (px-py mix for the oxo atom), with a fully occupied dxy metal orbital (which should be only partially occupied in an octaedrical FeV). The third unpaired electron in the high potential heme sits in the dxy orbital. Thus, as expected, there is an overall electron transfer from the high potential heme dxy orbital to the low potential heme dxy orbital, which is clearly characterized by the QM/MM methods. To gain insight into the electron transfer, we proceed again by selectively turning on/off different atoms in the quantum mechanical region. We should emphasize that all panels in Figure 6 are representative of compound I, the only difference being the selection of the quantum region. The middle and bottom panels in Figure 6 indicate the spin density for the medium and small quantum regions when removing the high potential heme and Trp94 from the quantum region, respectively. In the middle panel, when deleting the high potential heme from

Formation of Cpd I creates a highly electron-deficient metal center capable of oxidizing many substrates. Using computational techniques combining quantum chemistry with molecular mechanics, we can monitor the evolution of the spin density and follow the electron delocalizations along this process. The results for the apo-enzymes shown in Figure 3 are consistent with the experimental knowledge for these systems. In CCP, the full third unpaired electron is localized, in the proximal Trp. In mKatg, the presence of the unpaired electron in the distal tryptophan might be responsible for the covalent adduct between this tryptophan and Tyr229. In APX, several studies indicate the variability of the third unpaired electron.14 Interestingly, a covalent adduct between the distal Trp and the heme group has also been proposed,15 in agreement with the appearance of a small radical component in this residue. The excellent qualitative agreement of the theoretical spin density with the experimental observations in APX, CcP, and mKatG serves us to validate the computational approach to study the electron transfer pathway in the substrate bound APX and in the bacterial diheme cytochrome C peroxidase. The results in Figure 4 are consistent with the ascorbate oxidation by compound I. Oxidation starts along the O-O bond breaking and water dissociation. Because of the fast electron reorganization, any transfer of porphyrin spin density to the metal center is readily filled by electrons from the substrate, that is, the nuclear reaction coordinate is unable to trap the electron transfer pathway. Thus, we proceeded by modifying the quantum region to eliminate the possibility of an electron transfer from the substrate. Panels A and B in Figure 5 have the same geometry and only differ in the quantum region specifications. They clearly support the existence of a direct electronic pathway connecting the iron, the porphyrin π orbitals, and the propionate lone pairs to the ascorbate substrate, as suggested in the crystal structures. Importantly, the electron pathway in APX gets activated by the presence of the ascorbate ligand. When computationally mutating the ascorbate by neutralizing its negative charge, the propionate spin density disappears; the energy level of the propionate lone pairs is highly dependent on its electrostatic environment. Thus, the presence of the negatively charged substrate raises the energy of the propionate lone pairs and activates the electron transfer pathway. As mentioned before, in absence of substrate, mutations affecting the propionate screening affect the nature of the protein radical (porphyrin/ tryptophan radical). Thus, it seems like a delicate balance might tune the orbitals of the propionates, of the porphyrin, and of those residues in the active site vicinity, an effect capable of small shifts in the orbital energy and reorganization of the unpaired electron. Eliminating the substrate from the quantum

13464 J. Phys. Chem. B, Vol. 112, No. 42, 2008 region, for example, induces more Cpd I character to the complex, as seen when comparing both panels in Figure 5; there is a larger spin delocalization, and the iron spin has now a larger dxz-dyz mixing. Thus, the nature of the description of the substrate (quantum or classic) has considerable effects on the orbital levels of the porphyrin and metal center. When applying the same technique to the diheme cytochrome C peroxidase from Pseudomonas Nautica 617, we observe similar results to those of the APX system. As seen in Figure 6, Cpd I is capable of oxidizing the high potential electrontransferring heme, which accounts for the total third unpaired electron. By altering the quantum mechanical region, we can track the spin delocalization and obtain an electron transfer pathway including the bridging Trp94 and the propionate lone pairs. As seen in Figure 2, the activation process for this enzyme involves a large rearrangement of this tryptophan and the propionate groups, consistent with our electron transfer pathway model. Thus, the activation process seems to be the lock of the electron transfer circuit. Conclusion By means of computational quantum chemistry, together with available crystallographic structures, we have obtained a direct evidence for the involvement of the heme propionates in the electron transfer pathway to the metal center in APX and CcP. This mechanism, which we call the propionate e-pathway, could represent a general motif for electron transfer in/out of the heme group. Interestingly, several other diheme enzymes present a close contact between the propionate groups; probably the most notable of them is cytochrome C Oxidase. In addition, in many proteins the porphyrin groups are accessible to solvent, facilitating the electrostatic tuning of the propionate environment by ligand and other protein interactions. Future work using electron dynamics techniques on a finite basis set obtained by means of QM/MM methods will address a more quantitative view of the mechanism. Acknowledgment. The Spanish MEC/CTQ2006-10262/BQU project and the Barcelona Supercomputing Center are acknowledged for financial and computational support. Supporting Information Available: Figure showing the spin density for three different snapshots (first, middle and last) along 1 ns of molecular dynamics for ascorbate peroxidase, and another showing the electron transfer pathway obtained by the Harlem program for the APX and bCcP systems. This material is available free of charge via the Internet at http://pubs.acs.org.

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