J. Phys. Chem. 1996, 100, 6819-6824
6819
Theoretical Evidence of Electrophilic Superoxides in Models of Oxyhemocyanin/ Oxytyrosinase Active Sites. Influence of the Ligand’s Arrangement H. Getlicherman,† C. Giessner-Prettre,*,† and J. Maddaluno‡ Laboratoire de Chimie Organique The´ orique, URA 506 CNRS, UniVersite´ P. & M. Curie (Paris VI), Boite 137-4, place Jussieu-75252 Paris Cedex 05, France, and Laboratoire de Chimie Organique, URA 464 CNRS, UniVersite´ de Rouen and IRCOF-76821 Mont St Aignan Cedex, France ReceiVed: June 16, 1995; In Final Form: September 11, 1995X
The most widely accepted mechanism for the molecular dioxygen bioactivation by dicopper monooxidases, or their biomimetic models, in arene oxidation relies on a direct electrophilic oxygen transfer from an intermediate µ-η2:η2 peroxo dicopper complex into a substrate C-H bond. However, the electrophilic character of the activated form of dioxygen in such complexes has never been established. Extended Hu¨ckel calculations on a set of (Cu+(Im-H)3)2-O2 complexes, taken as model of oxyhemocyanin and oxytyrosinase active sites, are reported for both eclipsed and staggered arrangements of the imidazole ligands. The results obtained indicate such two conformations to be isoenergetic. In both situations, the molecular electrostatic potential maps show two striking features: (i) the plane perpendicular to the O-O bond and containing the two metal ions exhibit, as previously shown for the staggered situation, two nucleophilic potential wells; (ii) two strongly positive regions extend from the oxygens along the O-O axis. This last directional characteristic, not found in an isolated superoxo anion nor in molecular dioxygen, is the first direct evidence of the electrophilic character appearing for dioxygen binding on dicopper complexes. This feature is particularly striking considering the calculated charge value of -0.6 e on each oxygen atom. Variations in the results obtained for the staggered/eclipsed conformations of the ligands are discussed in relation with the differences between hemocyanin and tyrosinase as well as the various oxidative behaviors of their biomimetic synthetic models.
Introduction Despite a high degree of sequence homology for the domain of the protein surrounding their active site and closely similar spectral features of their oxy forms, the two widely spread dicopper proteins tyrosinase and hemocyanin exhibit very different activities.1-3 The former of these enzymes catalyzes a selective orthooxidation of phenolic structures while the latter one is a dioxygen-transporter protein encountered in several mollusks and arthropods hemolymph. Despite the long-lasting interest it rises, the origin of this difference of behavior has not been elucidated yet. In this perspective, and in regard to the fundamental aspect of dioxygen bioactivation mechanism underlying this problem, many efforts have been dedicated to the structural characterization of the oxy forms of the enzymes themselves. Simultaneously, significant progress has been achieved on simplified synthetic model complexes2,4 which have also been the subjects of a few theoretical investigations in the recent years.5 Many of the studied dioxygen-binding models can oxidize their own endogenous ligands,6 while only a limited number is able to process, as tyrosinase does, exogenous substrates.4g,6e,g,7 In the latter case, two types of oxidation products can be obtained. One features the expected (“monooxygenase-like”) benzoquinone structure while the other derives from oxidative radical coupling reactions. For the details of these reactions remain, in all cases, unclear, new mechanistic evidences are required before a conclusive catalytic cycle may be proposed.4f,g,7a,8 The most widely accepted chemical pathway calls for a direct electrophilic attack of an R-C-H bond in the phenoxo appendage by the putative µ-η2:η2 peroxide. Much experimental evidence in favor of such †
Universite´ P. & M. Curie (Paris VI). Universite´ de Rouen. X Abstract published in AdVance ACS Abstracts, March 15, 1996. ‡
0022-3654/96/20100-6819$12.00/0
an hypothesis has been gathered through years,6f,g and the importance of a good positioning of the peroxo group with respect to the aryl nucleus in enhancing the reactivity has been pointed out.4f,6f The only theoretical approach to this specific problem published recently consists of a set of X-R results, indicating a µ-η2:η2 peroxide to appear less nucleophilic than the corresponding cis or trans µ-1,2-peroxides.9 Thus, the possibilities for such an intermediate to behave as a strong electrophile remain to be established4g especially when considering that the electrostatic potential maps drawn for a model complex in the plane perpendicular to the O-O bond and containing the two copper cations exhibit negatiVe potential wells significantly deeper than in isolated dioxygen.10 The role of the arrangement of the ligands around the copper cations can also become, in the perspective of a sterically controlled reactivity, highly significant. However, only a limited number of crystallographic data concerning relevant complexes are available. The various X-ray studies published for two hemocyanins11 and one oxyhemocyanin12 all display a staggered arrangement of the ligands about the Cu-Cu direction. These results have been extended, on the bases of Raman spectroscopic studies, to oxyhemocyanins of three different origins.13 A comparable staggered arrangement has been observed in two synthetic models.4a,c By contrast, and as recently emphasized,11d an eclipsed conformation of the ligands at a dicopper site has been observed in zucchini ascorbate oxidase.14 Other eclipsed arrangements of the ligands have also been reported for a functional model of dopamine β-hydroxylase6j and two synthetic complexes which reversibly bind dioxygen.15 Ab initio quantum mechanical calculations16 on the first of these latter systems15a,b indicate dioxygen to bind in an η2:η2 mode close to that observed in all other characterized related dioxygen complexes.4a,c,e-h,6j,17 In relation with these results, dinuclear complexes studied by Casella et al.6g,18 ought to be considered, for they are able to © 1996 American Chemical Society
6820 J. Phys. Chem., Vol. 100, No. 16, 1996 SCHEME 1
oxidize exogenous phenolic compounds. The chemical structure of these complexes, for which no X-ray analysis has been published to our knowledge, most probably implies an eclipsed arrangement of the ligands. This set of results suggests that the geometrical arrangement of the ligands is of prime importance for the mimicking of the enzymatic activity and that eclipsed arrangements seem to favor oxidative behaviors toward C-H bonds. Since recent extended Hu¨ckel (EHT)4c and ab initio16 calculations on (Cu+(NH3)3)2-O2 also indicate that the staggered and eclipsed arrangements of the nitrogenous ligands lead to isoenergetic situations, it is clear that both types of conformations are to be considered. We thought that both problems of complexed-superoxide electrophilicity and its conformational variations might be tackled using an approach previously applied to a model of oxyhemocyanin active site.10 We thus undertook a study of the electronic/energetic characteristics of a relevant dicopper model complex and of its variations with the ligands arrangements. The molecular electrostatic potential maps in particular have been computed in different planes in order to examine the possible existence of an anisotropic electrophilic area/region close by the complexed dioxygen. Computational Procedures and Input As in our previous study,10 we have selected the (Cu+(ImH)3)2-O2 model complex for these calculations. Imidazole (ImH) is a fair model for histidine and is to be used here since recent results tend to show that the exact nature of the ligand is of prime importance for the fine-tuning of the electronic properties of the active site of the enzymes and complexes of concern.19 This choice leads to a large-size system (288 electrons) for which we had to resort to the extended Hu¨ckel theory (EHT). Since EHT is known to give unrealistic bond lengths, distances between bonded atoms have been, as in our previous study,10 kept constant in all computations. With this precaution this method appeared adequate for this type of problem. The computations have been limited to the threshold staggered (St) and eclipsed (Ec) arrangements of the Im-H around the copper cations. These conformations are detailed in Scheme 1 using an extended representation of a previously published scheme.10 Intermolecular bond distances and valence angles used as input for the two Im-H ligands conformational arrangements (Scheme 1) have been taken as identical. Such a procedure is expected to avoid possible artifacts due to minor variations of geometrical parameters. The consistence of this
Getlicherman et al. approximation has been checked by comparing the data obtained from an ab initio optimization of the intermolecular parameters (plus the O-O distance) of the smaller (Cu+(NH3)3)2-O2 system.16 These computations give a 0.35 kcal/mol energy difference in favor of the staggered conformation and close geometrical parameters for both situations. The values retained for the intermolecular parameters of the arrangement for St and Ec situations have been (i) inspired from experimental data11c,d,12b,c and nonempirical calculations16 and (ii) in line with our previous study on St complexes.10 Both St and Ec conformers are represented in Scheme 1 and Figure 1. We have limited our study to arrangements in which the dioxygen bond is perpendicular to the Cu-Cu direction as indicated by theoretical results9a,10,20 and experimental data concerning oxyhemocyanins.12a,b,13 The value of the Cu-Cu and O-O distances on the one hand and of the N-Cu-Cu and N-Cu-Cu-N angles on the other were taken from our ab initio results16 on (Cu+(NH3)3)2-O2. The choice of theoretical parameters provides a geometry respecting the overall symmetry of the model system considered. However, for four of the Cu-N distance the value retained is 1.9875 Å, Viz. the average of the “short” values measured for hemocyanins.11c,d,12b Since both SCF and MP2 ab initio computations show that the Cu+-N distance is shorter when the ligand is an imidazole than when it is an ammonia (1.73 and 1.79 Å, respectively at the MP2 level)21 we thought that the experimental values would be more appropriate than the theoretical ones which were obtained for NH3. For the two remaining “remote” imidazoles labeled N1 and N1′, Cu-N is taken equal to 2.4 Å since both experimental11c,d,4c,e,12b,15c and theoretical16 data tend to show that in systems such as the one studied here two of the ligands are more distant from the metal cations. The most recent crystallographical data published on oxyhemocyanin have confirmed the longest Cu-N bond to be in this distance range.12 In addition, and as we shall see, our results depend only quantitatively on this value. The O-O bond has been taken, in all calculations, perpendicular to the Cu-N plane (R ) 90 in Scheme 1b,d) as indicated by experiment4a,c,e,12 and theory.10 The bond lengths and angles of the imidazole have been taken from neutron diffraction data.22 Finally and given these inputs, we have, as in our previous study10 allowed the torsion angles φs (about the short Cu-N bonds) and φ1 (about the long ones) of the imidazole planes to vary (Scheme 1a). The molecular electrostatic potential (MEP) maps are calculated using the approximations developed by Daul et al.23 Previous results have shown that such calculations: (i) provide a good match between the negative/positive areas in molecular electrostatic potential maps and the nucleophilic/electrophilic character of reaction sites in zeolites and organometallic complexes for instance;23,24 and (ii) are in reasonable agreement with the corresponding ab initio results as can be seen from Figures 1S and 2S of the supporting information as well as from maps of ref 23. Results and Discussion The energy values and the atomic net charges obtained for the eclipsed and staggered conformations of the (Cu+(Im-H)3)2O2 complex are reported in Table 1. Keeping φs ) φ1, the energy minimum is reached when the torsion angle is equal to 30° for both staggered and eclipsed situations. These conformations are taken hereafter as references and the energy of the staggered one is taken as zero. We see from the corresponding tabulated values (Table 1, entry 1) that a staggered arrangement for the imidazoles leads to a stabilization of ∼16 kcal/mol with respect to the corresponding eclipsed system. By constrast, no
Theoretical Evidence of Electrophilic Superoxides
J. Phys. Chem., Vol. 100, No. 16, 1996 6821
Figure 1. Molecular electrostatic potential maps of (Cu+(Im-H)3)2-O2 in the Cu-N plane (A and B) and in the plane containing the O-O bond and perpendicular to the Cu-Cu direction (C and D) (values in kcal/mol): A and C, staggered conformations; B and D, eclipsed conformations.
TABLE 1: Variations of the Energies (in kcal/mol) and Atomic Net Charges for the Staggered (St) and Eclipsed (Ec) Conformations of the (Cu+(Im-H)3)2-O2 Complexes St
Ec
entry
φsa
φ1b
lc (Å)
E
QCu
QO
E
QCu
QO
∆Ed
1 2 3e
30 30 30
30 90 90
0.00 0.00 0.20
-78787.9 -78787.7 -78785.6
+0.475 +0.475 +0.432 +0.515
-0.578 -0.578 -0.583 -0.583
-78772.3 -78787.3 -78789.3
+0.474 +0.474 +0.480
-0.578 -0.578 -0.586
+15.6 +0.4 -3.7
a Torsion angle about the short Cu-N “bonds”. b Torsion angle about the long Cu-N “bonds”. c Distance from the CuN plane to the dioxygen midbond. d ∆E ) E(Ec) - E(St). e l * 0.00 implies a symmetry loss in the St complex leading to different charges for the two Cu and O atoms.
difference is found for the copper and oxygen atomic net charges. If φ1 is then varied from 30 to 90°, the St and Ec arrangements become almost isoenergetic (entry 2) due to the 15 kcal/mol stabilization of the Ec form and a negligible 0.2 kcal/mol destabilization of the St conformer. This energy
lowering is likely to be due to both the decrease of obvious steric repulsions between the two “remote syn” imidazole rings and the improvement of their electrostatic interaction. Furthermore, if the dioxygen molecule is moved by 0.20 Å out of the CuO plane toward N1 and N2′ in the Ec case (Scheme 1), an
6822 J. Phys. Chem., Vol. 100, No. 16, 1996 additional stabilization (2 kcal/mol) occurs for Ec while the St form is further destabilized (2 kcal/mol). Interestingly, this last Ec arrangement leads to the oVerall most stable complex albeit it probably does not correspond to the most stable conformation because of the limitations of the present study. However it clearly indicates that eclipsed arrangements are to be considered along with staggered ones, in nice agreement with indications reported by Karlin and colleagues4e from EXAFS simulation on [Cu2(Me2Im)6O2)]2+ and with out own nonempirical theoretical results on the corresponding (Cu+(NH3)3)2-O2 model complexes.16 For our reference conformations (φs ) φ1 ) 30°) the St complex energy decreases while that of the Ec one increases when the two long Cu-N distances are further lengthened. This feature is not observed when φ1 ) 90°; in this case the energies of both complexes undergo parallel decreases. The stability of the Ec-type complexes is thus critically dependent upon φ1 value. This dependence is of course reinforced for shorter CuN1/N1′ distances. In Figure 1 are displayed the molecular electrostatic potential maps of the most stable St and Ec complexes for the conformations corresponding to entries 1 and 3 of Table 1, respectively. For the St arrangement, the isopotential curves drawn in the Cu-N plane (Figure 1A) are, as expected, similar to those obtained previously.10 From Figure 1B, we see that for the Ec conformation, the potential well located between the four short Cu-N bonds is similar to those of Figure 1A and that a somewhat deeper (-110 vs -69 kcal/mol) and definitely larger pit appears symmetrically with respect to the Cu-O plane. On the other hand it is clear from Figure 1, parts C and D, that in the plane containing the O-O bond and perpendicular to the Cu-Cu direction, the O-O axis exhibits strongly positive areas despite the Cu(I) f O2 charge transfer occurring upon complexation as can be seen from values of the atomic charges reported in Table 1 (each oxygen carries a formal charge of about -0.58 e). Thus, in the complex, the dioxygen entity is best described as a superoxide anion. For an isolated superoxide anion, we see from Figure 2A that the molecular electrostatic potential is this time strongly negative in all space directions, in agreement with the nucleophilic character of O2-. The isopotential map of O2 reported in Figure 2B is also strikingly different from those in Figure 1C, D. In the former, the positive regions extending along the O-O bond correspond to molecular electrostatic potential values
