12212
J. Phys. Chem. B 2001, 105, 12212-12220
First Principle Calculations for the Non-Heme Iron Centers of Lipoxygenases: Geometrical and Spectral Properties Tomasz Borowski,† Marcin Kro´ l,‡ Maksymilian Chruszcz,† and Ewa Brocławik*,§ Faculty of Chemistry, Jagiellonian UniVersity, Ingardena 3, 30-060 Krako´ w, Poland, Department of Biostatistics and Medicinal Informatics Collegium Medicum, Jagiellonian UniVersity, Kopernika 17, 31-501 Krako´ w, Poland, and Institute of Catalysis and Surface Chemistry, Polish Academy of Sciences, Niezapominajek, 30-239 Krako´ w, Poland ReceiVed: June 20, 2001; In Final Form: September 10, 2001
Lipoxygenases (LOs), which are non-heme-iron-containing enzymes, play a vital role in plant and mammalian organisms. Their active sites have been probed by various spectroscopic techniques both in resting (Fe2+) and active (Fe3+) forms. Several crystal structures have been reported for ferrous forms of LOs; nevertheless, many unresolved questions have still remained. In particular, subtle differences in the first coordination sphere of the iron center seem to be very important for their catalytic activity thus any information about details of these structures is of great value. In this report, we present results of first principle calculations for reliable models of ferrous and ferric active sites of LOs undertaken to resolve ambiguities in structure of both resting and active forms of the iron sites in lipoxygenases. Geometrical parameters of optimized models are compared with crystallographic and EXAFS data. Time-dependent density functional theory (TDDFT) results for spectroscopic states are confronted with the relevant experimental results to validate the models and to gain an insight into the electronic structure of ferric and ferrous active sites. Overall good agreement between the calculated and experimental positions of the absorption bands is found, and where possible, the sources of discrepancies are discussed.
I. Introduction Lipoxygenases (LOs) constitute a class of non-heme iron containing enzymes catalyzing dioxygenation of polyunsaturated fatty acids. LOs are found in plants and animals and have versatile biological functions.1,2 The products of plant LOs, linoleic and linolenic acid hydroperoxides, are precursors of species important in development, growth regulation, wound response and pest resistance. On the other hand, 5-dioxygenation of arachidonic acid produces an intermediate in the synthesis of potent inflammatory agents (leukotriens and lipoxins) in animal organisms. Several X-ray crystal structures of LOs in their resting forms have been reported. Boyington et al.3 and Minor et al.4 have resolved the structure of soybean lipoxygenase-1 (SLO-1) at 2.6 and 1.4 Å resolution, respectively. Gillmor et al.5 have reported a 2.4 Å resolution structure of rabbit lipoxygenase (15RLO) with a bound inhibitor. Skrzypczak-Jankun et al.6 have solved the structure of soybean lipoxygenase-3 (SLO-3). The overall structures of these plant and mammalian LOs are very similar, but their active sites differ in first coordination sphere. For example, in SLO-1 resolved by Minor et al. the active site consists of ferrous ion coordinated by three histidines, Cterminal isoleucine, one water molecule and a loosely bound asparagine. The active site of the 15-RLO reported by Gillmor comprises four histidines and the C-terminal isoleucine in the * Corresponding author. E-mail:
[email protected]. Fax: 48 12 634-05-15. † Faculty of Chemistry, Jagiellonian University. ‡ Department of Biostatistics and Medicinal Informatics Collegium Medicum, Jagiellonian University. § Institute of Catalysis and Surface Chemistry, Polish Academy of Sciences.
iron first coordination sphere. The geometry of both types of active sites was described as a distorted octahedral, with the sixth coordination site blocked by inhibitor for 15-RLO. These differences in the first coordination sphere of the site seem to play vital role for their activity as SLO-1 is much more efficient for hydrogen atom abstraction from pentadiene unit in fatty acids than 15-RLO. Thus a lot of work is still devoted to clarify the detailed structure of active sites in LOs and understand the mechanism of their functioning. The electronic structures of the resting ferrous forms of SLO-1 and 15-RLO have been studied by means of near-infrared circular dichroism (CD) and magnetic circular dichroism (MCD) techniques.7-10 Results of these studies supported by the spectral analysis of model complexes11 have revealed that native SLO-1 exists as a mixture of five (5C) and six-coordinated (6C) Fe2+ species. Authors have proposed the weak and flexible Asn694 ligand to be responsible for that equilibrium in SLO-1. 15-RLO, in which this weak Asn is replaced by a strong His ligand, exists in one, six-coordinated form. Ferric sites of LOs have been characterized by means of X-ray spectroscopy,12 EPR, and MCD techniques.13-15 These studies have shown that ferric SLO-1 and 15-RLO exist in six-coordinated form, and that most probably an OH ligand replaces the water which is the sixth ligand in the resting form. In addition, the purple forms of LOs, in which H2O ligand is believed to be replaced by the peroxide radical, have been studied by EPR spectroscopy along with the active (ferric) forms. So far, theoretical studies on ferrous and ferric sites in LOs were limited to the simplified analyses of the relationship between symmetry and electronic structure of these sites based on carefully parametrized ligand field theory. Even if they
10.1021/jp0123637 CCC: $20.00 © 2001 American Chemical Society Published on Web 11/06/2001
Non-Heme Iron Centers of Lipoxygenases provided very valuable information on the site structure and led to well-grounded hypotheses about the origin of differences between their spectral properties, first-principle quantum chemical modeling has still been missing. With this end in mind we have undertaken high level DFT calculations for extended models of ferrous and ferric forms of active sites in SLO-1 and 15-RLO which are presented in the next sections of this report. These two LOs have been selected due to the wealth of experimental data available concerning both electronic and geometrical structure, and because they represent two classes of LOs differing in the first coordination sphere of iron as well as in activity. Reliable models of active sites in LOs were constructed from appropriate crystal structures deposited in PDB databasis. Next they were optimized, and a series of restraint optimizations were performed in order to assess the strength of the most interesting iron-ligand bonds. The transition energies to the lowest excited states of ligand-field origin were computed by TDDFT method for ferrous forms of the sites to be compared with the results of magnetic circular dichroism spectroscopy. Also the absorption spectra were calculated for ferric forms of lipoxygenases for the excitation energy range between 0 and 4 eV and they were related to UV-vis absorption spectroscopy. In addition the absorption spectra for so-called purple forms of LOs were computed in order to check the validity of the proposed structure for these species. On the basis of quantum chemical calculations we have quantitatively confirmed flexibility of the weak asparagine ligand and its influence on electronic states of the iron site in SLO-1. In contrast, the histidine ligand, which in 15-RLO replaces Asn, appeared to be bounded significantly stronger. We have also proposed new insight into the role of the water-based hydroxyl ligand in ferric states. II. Methodology Models of Active Sites. Geometry of ferrous forms of active sites in lipoxygenases has been investigated with X-ray crystallography and EXAFS spectroscopy. Two crystal structures available for SLO-1 assume different coordination for the ferrous cation. In the structure solved by Boyington et al.3 the active site geometry may by described as a highly distorted octahedron with two empty sites. The four ligands coordinated to Fe2+ ion are three histidines (His499, His504, and His690) and the monodentate carboxylate group of the C-terminal isoleucine (Ile839). The SLO-1 structure of Minor et al.4 resolved to 1.4 Å resolution has additional water ligand occupying one of these empty sites. In both structures, the sixth ligand, asparagine (Asn694), is too far away to make a bond with iron. The distances to Asn carbonyl oxygen equal to 3.2 and 3.1 Å in both resolutions, respectively. Nonetheless, this ligand seems to be very important as it has been proposed that Asn shifts on substrate binding or active site oxidation. Indeed, Solomon et al.8 have proposed that Asn acts as the sixth ligand for Fe in SLO-1 with bound substrate on the basis of near-infrared MCD spectra. In 15-RLO three histidines (His361, His366, and His541) coordinate the ferrous ion with their -N atoms while His545 donates its δ-N atom. Similarly to SLO-1, the C-terminal isoleucine (Ile663) makes the fifth bond with ferrous ion by only one of its carboxylate oxygen atoms. As 15-RLO crystal structure has been solved with a bound inhibitor in the active site, no water molecule coordinated to Fe ion is reported in this structure. The most important difference between SLO-1 and 15-RLO is that in rabbit LO the mobile Asn ligand is replaced by His, which makes a strong coordination bond with Fe ion.
J. Phys. Chem. B, Vol. 105, No. 48, 2001 12213
Figure 1. Crystal structure of the active site in SLO-1 (1YGE) and 15-RLO (1LOX).
Actual models of the active sites used in this work have been built from crystal structures obtained from PDB database (PDB entries: 1YGE and 1LOX for SLO-1 and 15-RLO, respectively). Both crystal structure geometries are shown in Figure 1. In our models shown in Figures 2 and 3 amino acids in the iron first coordination sphere have been replaced by their most relevant parts. Histidines have been represented by imidazole rings, asparagine by a formamide molecule and the C-terminal carboxyl group of isoleucine by a formic anion. The sixth water ligand, absent in the crystal structure of 15-RLO, has been added to the model of this LO assuming the geometry similar to SLO-1 structure. The peroxide intermediate product supposed to coordinate iron in purple forms has been modeled by a CH3OO fragment (see Figure 3). Geometries of these models have been fully optimized. In addition, the effect of the active site geometry and the influence of charge distribution on surrounding protein onto the electronic transitions has also been probed for SLO-1. Two models have been used here. The geometry of the first one (Cr1) has been taken from crystal structure by Minor et al. (1YGE) where relevant parts of amino acids coordinating ferrous ion have been replaced by suitable fragments as described previously. Missing hydrogen atoms have been added assuming typical bond lengths and angles. The second model (Cr2) is the Cr1 one embedded in a large number of point charges representing the rest of the protein and solvating water. The positions and charges of the environment have been obtained as follows. The catalytic domain of SLO-1 has been solvated with water within 5 Å from the protein surface. Hydrogen atoms have been added to the protein and crystal water and their positions, together with the positions of added solvent, have been determined by 39 ps long simulated annealing. During the first 9 ps, the temperature was kept at 300 K and throughout the rest of the dynamics it was lowered linearly to 0 K. The dynamics has been concluded by 1000 step minimization. In all molecular mechanics calculations AMBER16 force field has been employed. Originally, atomic charges were taken from the AMBER force field. Next the charges on carbon atoms replaced by hydrogen atoms in DFT calculations have been set to zero. The charges on remaining atoms of donating amino acids have been scaled so that the net charge of a residue was equal to integer value (0 for histidines and asparagine, -1 for isoleucine). The change imposed by this scaling procedure was less than 0.01 e per atom.
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Figure 2. Optimized geometry of ferrous SLO-1 (5C) and 15-RLO models superimposed on the crystal (in light) structure.
Figure 3. Optimized geometry of ferric SLO-1 and purple SLO-1 models.
Quantum Chemical Calculations. Quantum chemical calculations presented in this work concerned the models of iron centers in LOs which were extended to be realistic enough to describe subtle differences in iron coordination. Thus quantum chemical methodology had to be a high level correlated one due to demands of intricate electronic structure of iron center and, at the same time, computationally modest to treat our explicit models. Our methodology of choice, time dependent density functional theory (TDDFT)17 has already proved to be a reliable and computationally efficient tool for calculating electronic transition energies as well as oscillator strengths. As it is based on the response theory, the ground state electronic density is the only quantity needed to calculate the excitation energies and their intensities. Qualitative assignment of the excited states is gained from one-electron promotions within molecular orbitals giving rise to the density change on excitation. As present formulation of TDDFT takes into account only states differing from the ground state by one-electron promotions there
are some limitations on its application. Nevertheless, TDDFT has been successfully applied to a wide range of organic,18-20 and inorganic,21-23 molecular systems with an accuracy of 0.30.4 eV. Its strength and limitations in dealing with transition metal open-shell molecules have recently been assessed by us24 and this study indicated that also for such highly open-shell systems TDDFT is a robust methodology. All DFT computations have employed the three parameter exchange-correlation functional (B3LYP) due to Becke.25 Two different basis sets have been employed. The first one, LanL2DZ basis by Wadt and Hay26,27 (denoted here as BS1) is a double-ζ basis set with effective core potential (ECP) replacing innershell electrons on Fe atom. For H, C, N, and O atoms the D95 basis set due to Dunning28 combined with this ECP basis has been employed. This basis set has been used in all geometry optimizations and electronic transition computations as it has proved to be good enough for calculating these properties.24 The second basis set (BS2) used only in TDDFT electronic
Non-Heme Iron Centers of Lipoxygenases
J. Phys. Chem. B, Vol. 105, No. 48, 2001 12215
TABLE 1: Experimental and Calculated Bond Lengths (in angstroms) for Ferrous Active Sites in SLO-1 experimental
optimized
distance
1YGEa
2SBLb
SLO-1(5C)
SLO-1(6C)
Fe-His 499 N2 Fe-His504 N2 Fe-His690 N2 Fe-Ile839 O1 Fe-Asn694 Oδ1 Fe-Wat842 O
2.23 2.26 2.21 2.40 3.05 2.56
2.27 2.22 2.25 2.16 3.16
2.166 2.141 2.125 2.208 3.050 2.059
2.182 2.187 2.145 2.233 2.220 2.172
a
Taken from ref 4. b Taken from ref 6.
TABLE 2: Experimental and Calculated Bond Lengths (in angstroms) for Ferrous Active Site in 15-RLO distance Fe-His366 N2 Fe-His361 N2 Fe-His541 N2 Fe-His545 Nδ1 Fe-Ile663 O1 Fe-Wat O a
exptl 1LOXa
calcd 15-RLO(6C)
2.17 2.08 2.22 2.28 2.30
2.210 2.190 2.189 2.218 2.148 2.229
Taken from ref 5.
excitation calculations consisted of TZV basis set by Ahlrichs29 supplemented by one set of f polarization functions with the exponent equal to 1.05 for Fe atom, and 6-31G(d) basis set for ligands. The geometry of the models has been optimized at the B3LYP/Lanl2DZ level of theory with none or only one constraint. The constrained optimization was necessary to model the 5C form of SLO-1 and to assess the ligand-Fe bond strengths (vide infra). All first principle computations have been done with Gaussian 98 (Revision A.9).30 Force field calculations have been carried out with Discover program by MSI.31 Molecular orbitals have been drawn with the Cerius232 program by MSI. III. Results and Discussion Geometrical Structure of the Sites. Starting from the crystal structure geometry, the models of resting (ferrous) forms have been fully optimized in their quintet ground states. This spin state of ferrous forms of LOs seems to be well established by spectroscopic experiments33 and magnetic susceptibility measurements.34 Experimental results are also supported by ligand field theory which for d6 Fe2+ ion in roughly octahedral environment imposed by ligands of moderate strength assumes high spin electronic structure (e.g., quintet state). Because the Asn694 ligand seems to be apt to change its position and two limiting geometries have been suggested by experiment, we have optimized the geometry of ferrous SLO-1 within two schemes. In the first one, unconstrained geometry optimization gave effective octahedral geometry with six ligands coordinated at typical bond lengths. This model will be denoted as SLO-1 (6C). The second one, SLO-1 (5C), has been optimized with the distance between Fe and oxygen of Asn kept at the experimental value (3.05 Å), and this constrained optimization led to the effective five-coordinated (5C) structure. In Figure 2 we have shown optimized structures of 5C SLO-1 and 15-RLO (in its unique 6C form) superimposed on the experimental ones. In Tables 1 and 2 the geometric parameters of the ferrous active sites have been gathered. Inspection of these results reveals that bond distances of the crystallographic and optimized models agree well with the exception of Fe-Wat842 distance. The length of this bond in crystal structure (1YGE) amounts to 2.56
TABLE 3: Bond Lengths (in angstroms) for Optimized Models of Ferric Active Sites in SLO-1 bond
SLO-1
purple SLO-1
Fe-His 499 N2 Fe-His504 N2 Fe-His690 N2 Fe-Ile839 O1 Fe-Asn694 Oδ1 Fe-OH 842 O/Fe-OOR
2.157 2.137 2.181 2.048 2.176 1.862
2.176 2.144 2.173 2.017 2.120 1.933
TABLE 4: Bond Lengths (in angstroms) for Optimized Models of Ferric Active Sites in 15-RLO distance
15-RLO
purple 15-RLO
Fe-His366 N2 Fe-His361 N2 Fe-His541 N2 Fe-His545 Nδ1 Fe-Ile663 O1 Fe-OH/Fe-OOR
2.163 2.179 2.223 2.191 2.049 1.853
2.160 2.202 2.204 2.162 2.025 1.923
Å, which is significantly larger than our result and larger than the usual Fe-H2O distance in small complexes (2.06-2.22 Å).35 The uncertainty in geometry of the active site in SLO-1 has been signaled by the authors of this structure in their original work4 and pursued further in their recent paper36 which appeared while our work has already been submitted. In this new refinement of the crystal structure of SLO-1 the Fe-Wat842 distance amounts to 2.11 Å and agrees well with data for small complexes and distances in our optimized models. Moreover, comparison of the bond lengths for SLO-1 (5C) and SLO-1 (6C) reveals that the Fe-H2O distance is the most sensitive to the shift in the position of Asn. The evidence of a water-based ligand in the ferric active site of SLO-1 has been given by Nelson.13 The geometry of the ferric site of SLO-1 has also been probed with EXAFS spectroscopy,12 with the results supporting six-coordinated iron center. In modeling the ferric forms of active sites, we have assumed after ref 13 that the oxidation causes a hydrogen atom subtraction from the water molecule coordinated to iron. Thus, OH group would be the sixth ligand in the coordination sphere of the ferric ion instead of a water molecule. After Solomon et al.,38 who proposed that the purple forms of LOs might consist of the complexes between the active site and the peroxide radical, we have modeled such complexes with water based ligand substituted by the CH3OO group. The geometry of the models of ferric active sites of LOs together with their purple forms has been optimized without any constraints in the sextet ground states. This spin state is consistent with EPR results for active and purple forms of LOs.14 Figure 3 shows the optimized structures of ferric and purple forms of SLO-1. The bond lengths for the optimum structures of ferric states are given in Tables 3 and 4 for SLO-1 and 15RLO, respectively. The only available information on experimental geometry for ferric active sites of LOs comes from the EXAFS and XANES study for SLO-1 reported by Scarrow et al.12 Their results support the 6C model of ferric SLO-1 with one of the ligands at the distance to Fe amounting to 1.88 ( 0.02 Å and five other with the mean bond length of 2.12 ( 0.01 Å. They have also proposed another possible model in which two ligands with donating oxygen atoms are placed at distances of 1.87 and 2.03 Å from central ion and four N-donating ligands with the mean distance to Fe of 2.14 Å. The bond lengths of the optimized model of ferric SLO-1 agree very well with these data. The Fe-OH and Fe-Ile839 distances of 1.862 and 2.048 Å correspond to the two oxygen-based ligands in the Scarrow et al.’s model, while the mean distance
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Borowski et al.
Figure 4. The results of the potential energy scans for selected Feligand bonds.
of remaining ligands amounts to 2.163 Å, which is very close to the experimental value of 2.14 Å. Because two ligands, namely, water and asparagine, show the tendency to make a relatively weak bond in ferrous form of SLO-1, we have performed additional series of restrained optimizations in which the distance between the iron and the donating atom has been scanned. The purpose of these calculations was to assess the strength of these Fe-ligand bonds and to understand the equilibrium between 5C and 6C forms of ferrous SLO-1. Such computations have been carried out for Asn ligand, His ligand replacing Asn in 15-RLO, and the water based ligand. Both ferrous and ferric forms of the active sites have been examined. For the ferric site the OH ligand has been used in place of the water molecule (vide supra). Results of these computations are shown in Figure 4. The most interesting outcome of these PES scans is that indeed the Asn ligand makes much weaker bond than His. The energetic penalty for shifting the ligand from its equilibrium distance to 3.05 Å (the crystal Fe- - -O(Asn) separation) in ferrous forms amounts for Asn only to 4.02 kcal/mol and for His to 7.40 kcal/mol. Thus, this energy change for Asn ligand is of magnitude of a typical hydrogen bond (3-5 kcal/mol) and definitely higher for His ligand. For ferric forms of active sites, the Fe-ligand bonds are significantly stronger. The energy penalty now amounts to 7.07 and 9.86 kcal/mol for SLO-1 and 15-RLO, respectively. Thus, these results strongly support previously drawn conclusions that Asn ligand is indeed the weak one, and may shift its position easily in ferrous form of SLO-1 which leads to the change from 5C to 6C geometry. The existence of only one (6C) form for ferric SLO-1 and ferric/ferrous 15-RLO is also well explained by these results. In the latter case, ligand-iron bonds are too strong to be compensated by hydrogen bonds. The bond between ferrous ion and the Wat842 ligand is apparently a weak one and the shift of water position between calculated equilibrium and the distance in 1YGE structure costs no more than 3 kcal/mol, which remains in line with uncertainty in experimental assignments. The change in strength and length of the Fe-oxygen (Wat842) bond upon oxidation is also quite noticeable. Qualitatively, the character of this ligand changes from neutral water to negatively charged OH group, and as a consequence, the bond is much stronger and significantly shortened which confirms assumed interpretation of modification in iron coordination sphere upon oxidation. Spectral Properties. Ferrous forms of SLO-1 and 15-RLO have been probed by magnetic circular dichroism (MCD)
Figure 5. The orbitals in the β spin manifold for ferrous (5C) form of SLO-1 relevant to interpretation of MCD transitions.
spectroscopy by Pavlosky et al.8,9 These authors used alcohols as perturbing agents and showed that large spectral changes upon addition of these perturbers to SLO-1 may be explained if one assumes the existence of two forms (5C and 6C) of SLO-1 active site. No such changes have been observed for 15-RLO, and its spectrum is consistent with the six-coordination. The spectra of 6C forms have been resolved into two peaks around 8600 and 10300 cm-1, whereas for the 5C form of SLO-1 features have been found below 5000 and around 10600 cm-1. The authors proposed the following interpretation of these data: the two observed transitions are of d-d character, and the states involved are the lowest component of the symmetry split 5T2g ground state and the two components of the 5Eg excited state. The effective five coordination means bigger axial distortion and manifests in a large splitting of the two components of 5Eg state (∆5Eg). To confirm this interpretation we have calculated vertical excitation energies for the ferrous forms of the models studied here within the spin unrestricted TDDFT scheme. Two different basis sets described previously were employed to assess the basis set dependence of the results. For SLO-1 we have considered four models of ferrous active site. Two of them (5C and 6C) have been optimized, and two other have been based on crystal structure geometry of the SLO-1 active site (Cr1 and Cr2). In Figure 5 the most relevant molecular orbitals for spin minority manifold supplemented by orbital energy diagram for the SLO-1 (5C) are depicted. The analysis of one-electron promotions contributing to selected excitations gives the following TDDFT assignments. The two transitions in the region of 10000 cm-1 originate from one-electron promotions from 88β to 91β and 98β (with a small component of 88β f101β) orbital. Inspection of Figure 5 shows that these transitions are indeed of ligandfield origin with a small metal-to-ligand contribution. In Table 5 the experimental results are compared with these from TDDFT computations. The most noteworthy result, however, is that for the vacuum optimized models the experimental values of ∆5Eg are very well reproduced while the absolute excitation energies lie 1500-4000 cm-1 too high in energy. The results obtained for the experimental geometry (Cr1 model) are much closer to experiment with regard to absolute values of energy, but the splitting of the components of the 5Eg state is poorly reproduced compared to the vacuum model. The excitation energies computed with the larger basis set (BS2) are somewhat smaller,
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TABLE 5: Excitation Energies (∆E) and Low Symmetry Split of 5Eg Excited State (∆5Eg) for Ferrous Form of Active Sites in Lipoxygenases [cm-1] SLO-1 (5C) exptla
TDDFT (opt) /BS1 /BS2
15-RLO (6C)
∆5Eg
∆E
∆5Eg
∆E
∆5Eg
