What is the Real Nature of Ferrous Soybean ... - ACS Publications

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What is the Real Nature of Ferrous Soybean Lipoxygenase-1? A New Two-Conformation Model Based on Combined ONIOM(DFT:MM) and Multireference Configuration Interaction Characterization Hajime Hirao† and Keiji Morokuma*,†,‡ †

Fukui Institute for Fundamental Chemistry, Kyoto University, 34-4 Takano Nishihiraki-cho, Sakyo, Kyoto 606-8103, Japan, and Cherry L. Emerson Center for Scientific Computation and Department of Chemistry, Emory University, Atlanta, Georgia 30322

‡

ABSTRACT The geometric and spectral features of the ferrous resting state of soybean lipoxygenase-1 (SLO-1) have remained puzzling. We have theoretically characterized ferrous SLO-1 by means of the ONIOM(DFT:MM), TDDFT, and CASSCF/SORCI methods, taking explicitly into account the effect of the protein environment. Two conformations found theoretically in this study, Conf-A and Conf-B, have almost equal stability but have quite different geometries, with short and long Fe-O694 distances, respectively. While neither of the geometries agreed well with the crystal structure of the enzyme, an averaged geometry showed excellent agreement. Therefore, we propose that the crystal structure reflects a mixture of these two conformations. The calculated circular dichroism (CD) spectra for Conf-A and Conf-B were found to agree well with the two experimental spectra obtained previously for “six-coordinate” and “five-coordinate” forms of ferrous SLO-1, respectively. SECTION Biophysical Chemistry

oybean lipoxygenase-1 (SLO-1) is a nonheme iron enzyme that oxidizes linoleic and linolenic acids to yield the corresponding hydroperoxides.1,2 SLO-1 has served as a model for dissecting the structure and function of lipoxygenases, which are ubiquitously distributed in plant and animal kingdoms.3 Past studies have demonstrated that SLO1 exhibits a variety of intriguing features and phenomena, including a unique coordination sphere around the Fe(II) site. Boyington et al. solved an X-ray structure of SLO-1 at 2.6 Å resolution and suggested that the iron center should be fourcoordinate.4 Minor et al., however, concluded from a crystal structure determined at 1.4 Å resolution (PDB code 1YGE) that the iron center has six ligands, including Asn694 and water.5 The Od1 atom (henceforth O694) of Asn694 was properly directed to Fe, but the relatively long Fe-O694 distance (3.05 Å) indicated that this residue serves as a rather weak ligand. They further refined the structure (PDB code 1F8N) and again observed a relatively long Fe-O694 distance (2.87 Å).6 Crystallographic studies have therefore converged onto the view that ferrous SLO-1 has six ligands, with one of the ligands, Asn694, being only loosely bound. However, the circular dichroism (CD) and magnetic circular dichroism (MCD) studies done by Pavlosky et al. revealed that ferrous SLO-1 exists as an almost equal mixture of two distinct forms, which are most probably five-coordinate and six-coordinate forms.7-9 The theoretical energy scan calculations performed by Borowski et al. for an active-site model (without protein

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r 2010 American Chemical Society

environment) at the B3LYP/Lanl2DZ level showed that the energy increase with increasing Fe-O694 distance is less rapid than that for other Fe-ligand distances, and also, the calculated TDDFT d-d transition energies showed reasonable correlation with the experimental ones.10 In their calculations, however, there was a single energy-minimum state, which had a short Fe-O694 distance (2.22 Å), as opposed to the long distance in X-ray structures. Lehnert and Solomon carried out B3LYP calculations on a simpler active-site model and concluded that the weak Fe-O694 bond is due to a sideways, tilted geometry of Asn694 imposed in the protein environment.11 Previous experimental and theoretical results do not seem to be consistent with each other. To resolve this issue, we performed ONIOM(B3LYP/[SDD(Fe),6-31G*(rest):AMBER] calculations12 on the SLO-1 enzyme for the quintet ground state with Gaussian 09,13 taking explicitly into account the effect of the enzymatic environment on the active site. Furthermore, TDDFT and CASSCF/SORCI excited-state calculations were performed in conjunction with the TZVP (for Fe, O, and N) and SV(P) (for C and H) basis sets to derive CD spectra. For multireference calculations, at first, state-averaged CASSCF(8,7) calculations were done considering five roots (5SA-CASSCF(8,7); see Figure S1 in the Supporting Received Date: February 3, 2010 Accepted Date: February 16, 2010 Published on Web Date: February 19, 2010

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DOI: 10.1021/jz1001456 |J. Phys. Chem. Lett. 2010, 1, 901–906

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Figure 1. ONIOM optimized structures of (a) Conf-A and (b) Conf-B. For specific geometric parameters, see Table 1. Scheme 1. (a) Two QM Regions Used for QM/MM Calculationsa; (b) Two Water Conformations Considered

perturbation treatment, and the multireference Davidson correction was applied. The excited-state calculations were done with ORCA, ver. 2.6.35.15 An X-ray structure of SLO-1 (PDB code 1F8N, 1.4 Å resolution) was used as a starting point for our ONIOM QM:MM modeling. As shown in Scheme 1a, QM regions were defined so that key atoms around the iron were treated quantum mechanically. The larger 68 atom Model-1 was used for ONIOM geometry optimizations for the enzyme system containing a total of 14043 atoms, while the further truncated 41 atom model Model-2 was used for excited-state calculations. From the X-ray structure that contains heavy atoms only, it is not clear how the O-H bond of the water ligand (w841) is oriented. Therefore, we considered two possible conformations, Conf-A and Conf-B (Scheme 1b). In either conformation, one of the hydrogen atoms in the water ligand is H-bonded to the carboxylate part of Ile839. The two conformations differ in principle only in the orientation of the other O-H bond of the same water molecule. In Conf-A, it points to the side of His504, while in Conf-B, it points in the opposite direction, namely, to the side of Asn694. Note that the previously investigated conformation appears to correspond to Conf-A.10 More methodological details can be found in the Supporting Information. Figure 1 displays the ONIOM optimized geometries for Conf-A and Conf-B. Specific geometric parameters for each conformation are summarized in Table 1. While the difference between the two conformations, in principle, appears to exist only in the orientation of the water O-H bond (Scheme 1b), it turns out that this difference causes many other substantial geometric differences. In particular, major differences arise in the Fe-O694 distance and the Ow-Fe-N690 bond angle. In Conf-A, the Fe-O694 distance was short (2.39 Å), and the bond angle Ow-Fe-N690 was close to 180° (176.9°). By contrast, Conf-B had a significantly longer Fe-O694 distance (3.46 Å) and a much smaller Ow-Fe-N690 angle (140.0°). The major source of these differences between Conf-A and Conf-B is the different efficiency of the H-bonding interaction between w841 and Asn694 in these conformations. In ConfA, the O-H bond of w841 points away from Asn694, and

a

Wavy lines represent QM-MM boundaries. The residue numbering is based on the PDB file 1F8N.6

Information). Using the converged 5SA-CASSCF wave functions as reference states, SORCI calculations14 were further performed to incorporate the dynamical electronic correlation effect employing the resolution of the identity (RI) approximation. For the RI approximation, the TZV/C (for Fe, O, and N) and SV/C (for C and H) auxiliary basis sets were used, and for SORCI calculations, threshold values of Tpre = 10-4, Tsel =10-6, and Tnat =10-5 were used. Minor contributions from unselected configurations were evaluated by a


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Table 1. Key Geometric Parameters for the Two Calculated Structures and Three X-ray Structuresa r(Fe-Ow)

r(Fe-N499)

r(Fe-N504)

r(Fe-N690)

r(Fe-O839)

r(Fe-O694)

Ow-Fe-N690

Theory Conf-A

2.21

2.22

2.30

2.23

2.06

2.39

176.9

Conf-B

2.10

2.21

2.16

2.17

2.12

3.46

140.0

average

2.15

2.22

2.23

2.20

2.09

2.93

158.5

Experiment WT (1YGE)

2.56

2.23

2.26

2.21

2.40

3.05

154.0

WT (1F8N)

2.11

2.21/2.24b

2.34

2.29

2.28

2.87

157.4

Q697E (1FGR)

2.26

2.12/2.41b

2.11

2.24

2.47

3.41

140.9

a

Bond distances are in Å, and angles are in degrees. b Values for two different conformations.

Figure 2. CD spectra obtained by (a) theory and (b) experiment. The experimental spectra were adapted with permission from the American Chemical Society.8

these parts thus cannot form a H-bond with each other. Moreover, the lone-pair electrons of Ow and O694 will exert a repulsive force between the two sites. Consequently, the Ow-Fe-N690 part has an almost collinear arrangement. On the other hand, a hydrogen atom of Conf-B points toward the side of Asn694. Therefore, w841 is attracted and tilted toward Asn694 due to a favorable H-bond interaction, which makes the Ow-Fe-N690 angle smaller. Importantly, for this H-bonding interaction, a longer Fe-O694 distance is favorable


because the O-H bond of w841 and O694 can then have a proper, nearly collinear arrangement. We have seen that Conf-A and Conf-B show distinct differences in coordination geometries, in particular, in r(Fe-O694) and Ow-Fe-N690. It appears, however, that r(Fe-O694) for Conf-A is too short compared with the distance for wild-type enzyme geometries (3.05 Å for 1YGE or 2.87 Å for 1F8N), while r(Fe-O694) is too long for Conf-B. Also, Ow-Fe-N690 is too large for Conf-A, while it is too small for

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Table 2. Relative Energies (in kcal/mol) of Two Conformations at Various Levels of Theory Conf-A

Conf-B

ONIOM-ME

5.9

0.0

ONIOM-ME(þD)//MEa

3.1

0.0

ONIOM-EE//ME

3.8

0.0

ONIOM-EE(þD)//MEa

1.0

0.0

a

Corrected for dispersion energy by B3LYP-D.

Conf-B. These deviations led us to a working hypothesis that the X-ray structure reflects a mixture of Conf-A and Conf-B. To verify this hypothesis, we calculated averages of the geometric parameters for Conf-A and Conf-B and compared them with experimental values (Table 1). Interestingly, the averaged r(Fe-O694) and Ow-Fe-N690 values, 2.93 Å and 158.5°, respectively, agree fairly well with the experimental values for wild-type SLO-1, thus supporting our hypothesis. Another interesting comparison can be made with the X-ray structure for the Q697E mutant, which exhibits geometric parameters quite different from those of the wild-type enzyme (Table 1).6 We find that the experimental geometric parameters for the Q697E mutant agree very well with the theoretical values for Conf-B. This, in turn, suggests that the X-ray structure for this mutant has a dominant contribution from Conf-B, although the reason for this is not clear at this point. Table 2 lists the relative energies of Conf-A and Conf-B calculated using Model-1 with several methods. In all cases, Conf-B is slightly more stable than Conf-A. ONIOM-ME predicts that Conf-B is more stable than Conf-A by 5.9 kcal/mol, and single-point B3LYP-D dispersion energy correction for the QM region at the ONIOM-ME optimized geometries reduces the energy difference between Conf-A and Conf-B to 3.1 kcal/ mol.16 This is because as r(Fe-O694) decreases, the dispersion interaction between Asn694 and the rest of the QM region becomes larger. With the ONIOM-EE model, the energy difference was even smaller by 2.1 kcal/mol. These calculations demonstrate that Conf-A and Conf-B have similar stability, with the latter being slightly more stable. Thus, ConfA and Conf-B can coexist. In order to gain more insight into the significance of the two conformations, CD spectra have been calculated for ConfA and Conf-B at the ONIOM optimized geometries by using the TDDFT and CASSCF/SORCI methods and a reduced QM model (Model-2). Figure 2 shows the theoretically calculated CD spectra, along with the experimental ones. Overall, TDDFTand SORCI yielded similar spectral patterns, although the transition energies obtained by SORCI agreed better with the experimental values than those obtained by TDDFT. Both spectra obtained by TDDFTand SORCI for Conf-A had positive and negative peaks, while the two peaks for Conf-B were both positive (Figure 2a). The sign of the rotatory strength is determined by the angle between the electric and magnetic transition dipole moment vectors. This angle is smaller than 90°, and the rotatory strength is positive, except for the case of the higher-energy transition in Conf-A. It is clearly seen in Figure 2 that the spectral pattern for Conf-A agrees very well with that for the “six-coordinate” form obtained experimentally (Figure 2b). Moreover, the spectra for Conf-B agreed very well


Figure 3. Energy diagrams of DFT β-MOs for Conf-A and Conf-B.

with the experimental spectra for the “five-coordinate” form. Hence, our results suggest that Conf-A and Conf-B have a oneto-one correspondence to the “six-coordinate” and the “fivecoordinate” form, respectively. To understand the differences in the electronic excitation pattern between Conf-A and Conf-B, we further analyzed molecular orbitals (MOs). The system has a quintet ground state with all d-type R-MOs occupied and only one β-MO occupied. Figure 3 shows the d-type β-MOs for Conf-A and Conf-B. In Conf-A, a dyz-type MO, which is distributed on the plane defined by Fe, Asn694, and w841, is the most stable and thus occupied. The lower-energy peak of the experimentally observed d-d transition in Conf-A (Figure 2a) is therefore associated with an electronic excitation from the dyz-type MO to the dz2-type MO, and the higher-energy transition is due to an excitation from the dyz-type MO to the dx2-y2-type MO. The energy levels of d MOs for Conf-B were raised relative to those for Conf-A, except for the case of dz2 (Figure 3). The energy difference between the dz2 orbital and the lowest-energy d orbital for Conf-B is thus smaller than that for Conf-A. This is because Asn694 on the z-axis is far away from Fe and the ligand field in this direction is weaker in Conf-B. This results in lower excitation energy for the transition to dz2 in Conf-B than that in Conf-A, which agrees with the experimental trend, as seen in Figure 2. Interestingly, the most stable d MO for Conf-B is dxz, instead of dyz. The interchange of the d MOs occurred in

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the spectral pattern for Conf-A having r(Fe-O694) = 3.0 Å did not agree with the spectra for the “five-coordinate” form. Likewise, the artificial Conf-B geometry having a short Fe-O694 distance (2.4 Å) did not agree with the experimental spectra for the “ six-coordinate” form (Figure 4b). Therefore, participation of Conf-A having a long r(Fe-O694) or that of Conf-B having a short r(Fe-O694) can be ruled out. In summary, we have theoretically characterized the ferrous resting state of SLO-1 by means of the ONIOM(DFT:MM), TDDFT, and CASSCF/SORCI approaches. Two stable conformations, Conf-A and Conf-B, were examined and found to have similar stability but quite different geometric features. Conf-A has a short Fe-O694 distance, while Conf-B has a long Fe-O694 distance. It is proposed that the crystal structure for the enzyme reflects a mixture of these two conformations. The calculated CD spectra for Conf-A and Conf-B agreed well with the experimental spectra for putative “six-coordinate” and “five-coordinate” forms, respectively. Therefore, we propose that the two forms observed in the CD analysis are ConfA and Conf-B. We thus conclude that, in both the crystal for X-ray analysis and the solution for CD analysis, Conf-A and Conf-B coexist.

SUPPORTING INFORMATION AVAILABLE Complete ref 13, methodological details, active space, SORCI orbitals, and the coordinates for the QM atoms. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author: *To whom correspondence should be addressed. E-mail: morokuma@ fukui.kyoto-u.ac.jp. Figure 4. Comparison of SORCI CD spectra for (a) short/long Fe-O distance Conf-A geometries and (b) short/long Fe-O distance Conf-B geometries.

ACKNOWLEDGMENT H.H. thanks the FIFC fellowship. This work was supported in part by a CREST grant in the Area of High Performance Computing for Multiscale and Multiphysics Phenomena from the Japan Science and Technology Agency (JST). The computational resources at the Research Center for Computational Science at the Institute for Molecular Science (RCCS-IMS) and Academic Center for Computing and Media Studies of Kyoto University are gratefully acknowledged.

the two conformations probably because the distortion of w841 toward Asn694 in Conf-B aligns the lobe of the dyz orbital and the Fe-Ow bond. This makes the ligand field for dyz stronger and thus destabilizes this orbital in Conf-B compared to that in Conf-A. The electronic excitation patterns discussed here were found to hold in SORCI calculations as well (Table S1, Supporting Information). From the above analyses, it seems likely that Conf-A and Conf-B correspond to the “six-coordinate” and “five-coordinate” forms, respectively. Nevertheless, there remains another possibility that Conf-A with a short Fe-O694 distance and Conf-A with a long Fe-O694 distance correspond to the two forms observed in experiment, or Conf-B geometries with short and long Fe-O694 distances correspond to the two forms. In these cases, the orientation of the water ligand does not make a significant difference, but rather, the difference in the Fe-O694 distance matters. To examine the likelihood of this alternative, we performed additional SORCI calculations on the hypothetical states obtained by constrained geometry optimization calculations. In Conf-A and Conf-B, the distance was fixed at 3.0 and 2.4 Å, respectively; note that these states were less stable than the corresponding ground-state geometries. The results are shown in Figure 4. As Figure 4a shows,


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