A Density Functional Study of Possible Intermediates of the Reaction

8616

J. Phys. Chem. B 2001, 105, 8616-8628

A Density Functional Study of Possible Intermediates of the Reaction of Dioxygen Molecule with Non-Heme Iron Complexes. 1. N-Side versus O-Side Mechanism with Water-Free Model Maricel Torrent,† Koichi Mogi,† Harold Basch,†,‡ Djamaladdin G. Musaev,*,† and Keiji Morokuma*,† Cherry L. Emerson Center for Scientific Computation and Department of Chemistry, Emory UniVersity, Atlanta, Georgia 30322, and Department of Chemistry, Bar Ilan UniVersity, Ramat Gan, 52900, Israel ReceiVed: April 18, 2001; In Final Form: June 27, 2001

Mechanistic aspects of the biological activation of O2 catalyzed by methane monooxygenase (MMO) were investigated by using a hybrid density functional method. The reduced form of the metalloenzyme was modeled by cis-(H2O)(NH2)Fe(η2-HCOO)2Fe(NH2)(H2O), where the O2 molecule may coordinate the Fe centers from two different sides, the H2O-side and the NH2-side, leading to two different mechanisms, O-side and N-side pathways, respectively. Calculations show that both pathways proceed via similar intermediates. The energy profile for the reaction of O2 coming from the O-side, however, is more consistent with available experimental data than for the N-side. On the other hand, the N-side mechanism is thermodynamically more favorable. This study suggests that, if the protein backbone did not block the N-side, the O2 molecule would most likely approach the dinuclear iron center from this side rather than from the O-side. Several mixed-valence intermediates have been found during the reaction, including an FeII-FeIII mixed-valence species, P*, prior to formation of intermediate P, and a species similar to intermediate X in the analogous mechanism of Ribonucleotide Reductase, as well as an FeIII-FeIV mixed-valence species prior to formation of intermediate Q. Our theoretical findings give support to the idea that electrons do not need to be transferred by pairs in the studied diiron system. This is the first time that a structure for intermediate P* has been proposed in the literature.

I. Introduction Activation of the oxygen molecule for incorporation into organic substrates (oxygenase activity) catalyzed by metalloenzymes is a powerful process for utilizing hydrocarbons. 1 Nature has evolved a number of metalloenzymes to overcome the barrier of diatomic O-O bond cleavage, among which ironcontaining ones (such as the heme-containing cytochrome P4502) and non-heme proteins with carboxylate-bridging diiron sites3 play an important role in this transformation. Below, we will briefly discuss only the oxygenase activities of non-heme diiron proteins. Diiron species with terminal aquo/hydroxo/oxo ligands are implicated as key intermediates in several synthetic and biochemical catalytic cycles all of which involve O2 activation at some stage of the cycle (ranging from desaturation to oneelectron oxidation, peroxidation or ferroxidation, among others4). The selectivity and efficiency of these reactions and the mild reaction conditions in ViVo suggest a methodology distinct from harsh industrial processes, which usually require higher temperature and pressure, and traditional synthetic processes (even with chemical catalysts). Therefore, the development of biomimetic inorganic catalysts (i.e., complexes possessing the suitable active site and microenvironment to mimic the ability of non-heme enzymes to tune the reactivity of O2), has attracted the interest of the bioinorganic community in the past few years.5 Despite numerous positive developments in this area, there is † ‡

Cherry L. Emerson Center for Scientific Computation. Department of Chemistry.

still a dearth of structural and mechanistic information regarding the O2-activating dinuclear complexes. One of the important O2-activating diiron enzymes is methane monooxygenase (MMO). This metalloprotein, for which several biomimetic studies have been recently launched,5 is responsible for the conversion of inert methane into methanol. MMO is a classical monooxygenase6 where two reducing equivalents from NAD(P)H are employed to split the O-O bond of O2. One O atom is reduced to water by 2-electron reduction, while the second oxygen atom is incorporated into the substrate to yield an alcohol:

CH4 + O2 + NAD(P)H + H+ f CH3OH + NAD(P)+ + H2O (1) Efforts in the last 10 years to develop non-heme iron catalysts capable of modeling the chemistry of MMO have mostly given rise to complexes that generate alkoxyl or hydroxyl radicals.7 There are, however, a few exceptions that hint at the possibility that biomimetic alkane hydroxylation can actually be achieved. The further development of these promising systems and the design of other hydrocarbon oxidation catalysts of biotechnological interest require a better understanding of the actual enzyme. Computational studies may play an important role in elucidating the complexities experiments cannot resolve. The key initializing step in methanotrophic bacteria, Scheme 1, is postulated to be the fast reaction of O2 with the reduced form of the hydroxylase component of MMO (MMOHred), which contains two ferrous iron centers bridged by several

10.1021/jp0114743 CCC: $20.00 © 2001 American Chemical Society Published on Web 08/21/2001

Reaction of Dioxygen with Non-Heme Iron Complexes

J. Phys. Chem. B, Vol. 105, No. 36, 2001 8617

SCHEME 1: Postulated Intermediates in the Single Turnover Cycle of Diferrous MMOH in the Presence of MMOB after Rapid Mixing with O2 and Substrate Containing Solution

hydroxo/aquo and carboxylate ligands. This leads to the formation of a metastable enzyme-dioxygen complex (called compound O) that precedes the oxidation of the diferrous cluster. Intermediate O, in which dioxygen is proposed to bind to the enzyme but not to the dinuclear iron center, decays spontaneously to an intermediate designated as P*. Such a transient species is required by the observation8 that decay of the diferrous EPR signal is faster than the formation rate of the first optically detected intermediate, a diferric peroxy intermediate called P. Until recently, intermediates O and P* had been proposed based on indirect kinetic evidence.9 In a very recent study, Brazeau and Lipscomb10 have presented the first direct evidence for these species. Despite enormous efforts, however, there is currently no spectroscopic data available for species O and P* yet. Not much is known about the first detected intermediate of the catalytic cycle, peroxo diferric intermediate P, either. The molecular structure of intermediate P has been a focus of controversy between spectroscopists and is still a subject of current debate.11 What is unanimously accepted3c,d is that intermediate P further decays to produce intermediate Q. This intermediate is the first non-heme ferryl-oxo species to have been identified in biological systems and the only dinuclear Fe(IV) species known to date. In the catalytic cycle of MMO, Q is the intermediate believed to react with methane. So far, no evidence has been found for direct reaction of P with substrates. Meantime, the exact details of the mechanism leading P to Q remain still unknown. As seen from Scheme 1, two different substrates are activated along the reaction of MMO: first, dioxygen and then, methane (or alkane, in general). Our long-term goal is to study the mechanism of the full reaction

MMOHred + O2 f {FeIII(O22-)FeIII}P f {FeIV(O2-)2FeIV}Q {FeIV(O2-)2FeIV}Q + CH4 f {Fe2(µ-O)(µ-HOCH3)} f MMOHred + HOCH3 + H2O (1) In two recent papers,12 we have already studied the activation of methane by intermediate Q, i.e., the following part of the reaction 1

{FeIV(O2-)2FeIV}Q + CH4 f {Fe2(µ-O)(µ-HOCH3)}

(2)

In the present study we focus on the activation of O2 molecule, a nontrivial process which is supposed to take place right after the initializing step of the catalytic cycle, i.e., after the conversion of the resting state of the enzyme into the reduced active form.

MMOHred + O2 f {FeIII(O22-)FeIII}P f {FeIV(O2-)2FeIV}Q (3) Understanding of the O2 activation by MMO requires knowledge of, at least, two aspects. First, it would be desirable to know which coordination sites at the carboxylate-bridged catalytic center in the hydroxylase enzyme can be occupied by substrate molecules (O2) during turnover. So far, X-ray crystallographic studies of MMOH from Methylococcus capsulatus (Bath)13 have revealed four such positions (Scheme 2).14 It should be noted that three of these positions (sites 1-3 in Scheme 2) are located in the O-side of the iron cluster, i.e., the side opposite to where protein residues His147 and His246 lie (hereafter called N-side, see Scheme S1 in the Supporting Information for details). A fourth position is located in the N-side (site 4 in Scheme 2). Lippard and co-workers15 have found that all these positions can simultaneously accommodate methanol, water and DMSO; such species do not compete for the same coordination position. This information has direct implications on the mechanism of MMO, and suggests that O2 coordinates at the O-side of the iron cluster,16 site 3 in Scheme 2. Also, in a recent study it has been reported17 that hydrophobic species bind preferentially in preexisting cavities in MMOH and one of these cavities in the R-subunit has been located precisely in the O-side of the active center. Despite this, studies of (1) the O2 coordination from the N-side of the diiron center and (2) the mechanism of the O-O bond activation of the N-side coordinated O2 molecule alone with that of O-side coordinated O2 have a great importance from the biomodeling point of view. Here we will discuss the O-O bond activation for both the N-side and O-side coordinated O2 molecule. The second aspect to understand the mechanism of O2 activation by MMO concerns spin-crossing effects (more specifically, the so-called two-state reactivity18,19). Like many reactions involving the first-row transition metal atoms, O-O bond activation reaction on the diiron complexes may involve several lower-lying electronic states. As we already did in our previous work on C-H activation by MMO,12 in the present paper we study, at least, two spin surfaces, 9A and 11A (7A may also intervene in some specific points along the reaction). The purpose of the present paper is tri-fold. First we investigate the first steps of the reaction (O2 coordination) with special emphasis on additional intermediates preceding the formation of P. Possible identities of these additional intermediates include an enzyme-dioxygen Michaelis complex and a superoxo species. Second we explore aspects of the reactivity of MMO that might be interesting for future experimental and synthetic studies on improved biomimetic compounds having the same functionality of MMO. In this respect, a comparison of the N-side and O-side pathways from a computational point of view would be a first useful step to elucidate aspects that may affect the design of new biomimetic models. Finally, we analyze the features of dioxygen O-O bond cleavage in MMO by scrutinizing other intermediates besides the ones mentioned above (i.e., those experimentally observed or postulated). What is the exact sequence of intermediates between P and Q in the actual enzyme? And how does the reaction proceed from reactants to P? How can we connect the separate facts that are available from experimental data? The ultimate goal here is to bridge the gap between the isolated pieces of information we have from experiments so that a deeper insight into the enzymatic mechanisms can be gained. A final aspect worth exploring, which will not be fully addressed in the present work, is the nature of the O-O bond

8618 J. Phys. Chem. B, Vol. 105, No. 36, 2001 SCHEME 2: Schematic Representation of a Model of the MMOH Diiron Center Showing the Ligands Which Anchor Iron, and the Four Proposed “Open” Sites (Numbered 1-4) Which Are Expected to Be Available for Dioxygen and Substrate Binding

Torrent et al. in each case. Further details are given in the text below (see Results and Discussion). In these calculations a spin-unrestricted open-shell singledeterminant method, B3LYP,22 has been used in conjunction with the Stevens-Basch-Krauss (SBK) effective core potentials (ECP) and the standard 31G, CEP-31, and (8s8p6d/4s4p3d) basis sets for H, (C, O, and N), and Fe atoms, respectively.23 Full geometry optimizations have been carried out for all stationary points. The nature of these structures has been confirmed by performing vibrational frequency calculations (Nimag ) 0 for all equilibrium structures). All calculations have been performed by using Gaussian98.24 III. Results and Discussion

cleavage. Despite a general consensus regarding the formation of intermediate Q from intermediate P, it is not clear yet whether the O2 activation occurs homolytically or heterolytically. In the present study we have assumed an homolytic O2 activation. II. Computational Details To be consistent with our previous studies on methane activation,12 we have chosen here a similar model for the starting complex, MMOHred, cis-(H2O)(NH2)Fe(η2-HCOO)2Fe(NH2)(H2O), satisfying the restrictions imposed by experimentally available data: (1) It includes one histidine nitrogen (modeled by NH2) and one monodentate coordinated O-ligand (modeled by OH2) for each Fe center, and two bridging carboxylates (modeled by HCOO-) coordinated to the two Fe centers; (2) it has no net charge, consistent with the fact that the active site is buried in a low dielectric protein medium; and (3) imidazole rings of the His ligands are located cis to each other, i.e., two monodentate terminal carboxylates are cis to each other as well. One may be concerned with the adequacy of this small model system. However, we would like to emphasize the following. For conversion of methane to methanol by the Q complex, this small model was used by us to find the transition state for the hydrogen abstraction from methane to form a bound methyl hydroxyl intermediate and for the first time the transition state for reaction of methyl and hydroxyl ligands of the intermediate to form a methanol complex.12 Subsequently larger model systems were used by others20 to find exactly the same transition states and intermediate, proving that this small model system adequately described the essential feature of this metalloenzyme. Concerning the choice of the spin state for these systems, we have based our criterion on the best possible agreement between available experimental data and computational difficulty. According to spectroscopic studies of MMOHred the active center of this enzyme includes two ferromagnetically coupled FeII-centers, and has a total spin S ) 4 at the ground state.21 Therefore, we have considered our starting complex, MMOHred, as a ferromagnetically coupled high-spin species (2MS + 1 ) 9). Test calculations with multiplicity 2MS + 1 ) 7 have been also performed for MMOHred; our results indicate that the latter multiplicity is not the ground spin state for this system, in good agreement with the above assumption. Once MMOHred (9A) reacts with the ground-state O2 (3Σ), there are at least two possibilities: 2MS + 1 ) 7 and 2MS + 1 ) 11. Since these two multiplicities interplay with 2MS + 1 ) 9 along the reaction, we have calculated more than one electronic state per intermediate to ensure the correct multiplicity

This section is divided into four parts. First we focus on O2 coordination, i.e., on the steps of the reaction preceding formation of intermediate P. This part covers the characterization of intermediates that so far have not been experimentally isolated, which is the first main goal of the present work. (Before going through this part, it might be useful to some readers to look at Appendix I in the Supporting Information. It contains a brief discussion on the spin distribution on the active center along the reaction path necessary to better understand the results presented in this section). Second we investigate the suitability of the N-side vs the O-side focusing on the reaction steps from intermediate P to intermediate Q, i.e., specifically on O-O bond cleavage (O2 actiVation). Third we compare the two studied paths keeping in mind the initial spin analysis, and available experimental data. This is the second main goal of the present study. Finally we discuss an overview of the full mechanistic path (i.e., coordination + activation). Our working hypothesis is summarized in Scheme 3. This hypothesis will be taken as reference for discussing the calculated results. A. O2 Coordination: From Reactants to Intermediate P. Let us start our discussion with the active form of the metalloenzyme: MMOHred. The optimized geometry for this structure using the model cis-(H2O)(NH2)Fe(η2-HCOO)2Fe(NH2)(H2O) is shown in Figure 1. The structure of the ground 9A state possesses a quasi C symmetry consistent with the spin s analysis discussed in Appendix I, i.e., a structure with two almost identical FeII d6 centers (2MS + 1 ) 9), each with four alpha unpaired electrons (calculated spin population: Fe1 4.00, Fe2 4.00, Table 1). The ground triplet (3Σ) state of O2 has the configuration KK(σs)2(σs*)2(σp)2(πx)2(πy)2 (πx*)1(πy*)1, with one unpaired electron on each oxygen atom. Its open-shell “singlet “excited state (2MS + 1 ) 1) is calculated to be 29.2 kcal/mol higher than the ground triplet state (2MS + 1 ) 3). This triplet-singlet splitting, although not very accurate at this level of theory, is still in qualitative agreement with the experimental value of 22.5 kcal/ mol.25 The first step of the reaction is expected to be O2 coordination to the active site of the enzyme (Scheme 3). This could be a very complicated process, and may proceed with a notable energetic barrier required for reorganization of the active site and the close protein environment. Coordination of the O2 molecule to MMOHred may produce a weakly protein-bound complex, like compound O predicted by experimentalists.3 This type of protein-bound complexes, however, cannot be calculated for the present model lacking the protein environment. Instead, we have made several attempts to find the weakly bound O2MMOHred complex, [FeII(O2)FeII]. This complex may have several electronic states, among which we investigated only

Reaction of Dioxygen with Non-Heme Iron Complexes SCHEME 3: Flow of Intermediates, According to Our Working Hypothesis, for the Mechanism of the Biological Activation of Dioxygen by MMO with Special Emphasis on the Conversion from Intermediate P to Intermediate Q (Both Underlined)a

a Two paths are suggested for the homolytic activation of O : paths 2 A and B. Path C corresponds to heterolytic O-O bond cleavage.

those with 2MS + 1 ) 11, 9, and 7. We were not able to locate a minimum corresponding to [FeII(O2)FeII], and conclude that the weakly bound O2-MMOHred complex does not exist. During our search, full geometry optimization of the structure [FeII(O2)FeII] starting with initial Fe-O2 distances in the range 5.56.5 Å systematically led to complex P* shown in Figure 1. Compound P* can be described as a mixed-valence species, [FeII(O2)-FeIII], with end-on coordinated terminal O2-moiety. Three different states were calculated for this intermediate. The ground state of P* is an 11A state, as shown in Figure 2. The 9A state lies only 0.5 kcal/mol above the ground state. The spin distribution in the 9A state (Table 1) is different from that in the 11A only by the sign of the spin located on the O2-moiety, which is now antiparallel to the Fe spins. Since the coupling between the Fe centers and the O2-moiety is weak, this type of spin flip does not cause significant energy changes, which is consistent with the calculated small energy difference between 9A and 11A states. The 7A state corresponds to an unstable species, located 9.9 kcal/mol above the ground state. In the 7A state, in addition to the spin flip in the O2-fragment, two electrons of Fe2 are paired, which makes Fe2 a low-spin ferric iron. As usual, intra-atomic spin couplings are energy demanding processes (for Fe-complexes they usually require about 5-10 kcal/mol). This is consistent with the calculated energy gap between 7A and 11A states, as seen in Figure 2.

J. Phys. Chem. B, Vol. 105, No. 36, 2001 8619 Looking at the optimized molecular structures of the three electronic states of intermediate P*, Figure 1, it should be noted that the O-O distance (ca. 1.39 Å) is significantly longer than that calculated in the free O2 molecule (1.276 Å, Figure 1). The O2-moiety in complex P* corresponds indeed to a superoxo fragment, O2-, where the O-O bond has been partially activated upon one-electron transfer from the Fe atom on the right. (The experimental O-O distance26 in free O2- is 1.341 Å and at the current level of calculation, 1.416 Å). This one-electron reduction is also consistent with the spin analysis in Table 1. The diiron center of complex P* 11A has one FeII (with four unpaired electrons) and one FeIII (with five unpaired electrons). This is why this complex can be assigned to what experimentalists call intermediate P*: presumably an FeII-FeIII mixed-valence species with a superoxo moiety. To the best of our knowledge, this is the first time that a structure is proposed for intermediate P*. Experimentalists so far only postulated and have been unable to isolate and therefore characterize such a mixed-valence species. One should note that there also would be P*-like complexes with O2 approaching from the N-side, which we did not explore. Complex P* rearranges to the O-O cis-µ-1,2 type complex P shown in Figure 1, with a [FeIII(O2)2-FeIII] core. Such a µ-η1: η1-O2 mode of binding is in good agreement with recently characterized peroxo model complexes27 which possess a similar µ-η1:η1-O2 mode. Figure 1 shows the optimized geometry for intermediate P, labeled P-cis, with multiplicities 2MS + 1 ) 7, 9, and 11. The calculated O-O distance in the peroxo, O22-, fragment in these complexes is 1.47 Å. (The experimental O-O bond distance26 in O22- is 1.49 Å and the calculated value at the present level is 1.66 Å.) The ground-state intermediate P-cis (11A) is found to be more symmetric than P-cis (9A). The former has a nearly identical environment for the two iron centers, with approximately five unpaired electrons per iron (4.72 in Fe1 and 4.72 in Fe2), Table 1. The unfavorable intra-atomic coupling of a pair of electrons in P-cis (9A) causes a destabilization of 6.7 kcal/mol with respect to P-cis (11A), as shown in Figure 2. The second coupling of electrons increases the destabilization up to 12 kcal/mol for P-cis (7A). According to experiments on the actual enzyme, the first stable intermediate in the reaction of dioxygen with diferrous MMOH is intermediate P. Using the present model and level of calculation, complex P-cis (11A) is calculated to be 1.0 kcal/ mol higher in energy than complex P* (11A), which is a too small energy difference to draw any solid conclusion about relative stabilities in the actual (more complex) enzyme. One can only say that P* and P are very close in energy, probably with a small energy barrier between them. Experimental factors may well be responsible for having prevented experimentalist from detecting P* so far. From intermediate P, as shown in Scheme 3, the O-O bond undergoes cleavage and the reaction leads to intermediate Q through O2 activation. The O2 activation is discussed in detail in the next section. B. O2 Activation: N-side versus O-side for the Step P f Q. As mentioned in the Introduction, due to the arrangement of the protein ,only the O-side coordination of the incoming O2 molecule to the active site is experimentally feasible (Scheme S1). The investigation of the two distinct O2-coordination mechanisms, O-side and N-side pathways, may be relevant from the point of view of biomimetic studies and bioinorganic chemistry. Therefore, in this section we investigate the O2 coordination and activation from two different sides of the active

8620 J. Phys. Chem. B, Vol. 105, No. 36, 2001

Torrent et al.

Figure 1. Optimized geometries for the reactants and all intermediates involved in the first steps of the reaction (O2 coordination). Bond distances are in angstroms and torsional angles in degrees.

TABLE 1: Mulliken Atomic Spin Populations (in e) for All Reactants and Intermediates Involved in the First Steps of the Dioxygen Activation Reaction (i.e., O2 Coordination) structure

LnFe1

Fe2Ln

O2 7A P*_7 P-cis_7

O1

O2

1.00

1.00

oxidation statea

3.91 3.01

3.05 3.01

-0.37 -0.01

-0.59 -0.01

FeII FeIII (HS,LS) FeIII FeIII (LS,LS)

4.00 4.06 2.99

4.00 4.88 4.75

-0.34 0.05

-0.60 0.21

FeII FeII FeII FeIII (HS,HS) FeIII FeIII (LS,HS)

4.06 4.72

4.87 4.72

0.45 0.28

0.62 0.28

FeII FeIII (HS,HS) FeIII FeIII (HS,HS)

9A

reactant P*_9 P-cis_9 11A P*_11 P-cis_11

a HS ) high spin, LS ) low spin. Labels in parentheses correspond to the first and second iron, respectively.

site, N-side and O-side pathways. In the O2-coordination step (previous section) we have not differentiated between N-side and O-side because prior to formation of intermediate P, the

interaction between the coming O2 molecule and the enzyme is fairly weak. This step is not expected to affect dramatically the reactivity. Actual bond-breaking and bond-forming takes place beyond this point. N-Side. The geometries of the intermediates for the N-side pathway are shown in Figure 3. The energy profile for the N-side is depicted in Figure 4. Here we focus specifically on the P f Q step, with O2 coming from the N-side. The notation PQn (n ) 0, 1, 2) has been used to refer to the sequence of intermediates between P and Q. Hereafter and all throughout this section, the extension “_N” and “_O” indicates whether an intermediate belongs to the N-side or O-side pathway, respectively. Intermediates that are common to both pathways have no extension. In Table 2 we present the calculated Mulliken spin populations of LnFe1, Fe2Ln, O1, and O2 fragments in the optimized structures for the N-side. As mentioned previously, the first stable intermediate in the reaction of O2 with diferrous MMOH is intermediate P. In Figure 3 we present the optimized geometry for intermediate


Figure 2. Potential energy surface for the coordination of the O2 molecule to the active site of the enzyme. The energy of the reactants is taken as reference. Energies are in kcal/mol.

P-cis with multiplicities 2 MS + 1 ) 7, 9 and 11, similar to the ones shown in Figure 1 where O2 comes from the O-side. Here O2 comes from the N-side. The lowest electronic state for such an intermediate is 11A, Figure 4, with two high-spin iron centers. Again, this is consistent with our spin analysis summarized in Appendix I (in Supporting Information). At the present level of calculation, intermediate P-cis_N (9A) is 8.0 kcal/mol less stable than intermediate P-cis_N (11A), and intermediate P-cis_N (7A) is 7.3 kcal/mol less stable than P-cis_N (9A). As seen from Table 2, the most stable multiplicity is the one with a favorable HS FeIII-FeIII center: five spins on Fe1 (4.60) and five spins on Fe2 (4.60). The O-O distance is in the range of 1.46-1.47 Å. It should be noted from Figure 3 that P-cis_N (7A) and especially P-cis_N (11A) are quite symmetric, whereas P-cis_N (9A) has a much more asymmetric FeOOFe moiety. The spin densities for this intermediate in the three different multiplicities shown in Table 2 do also reveal a symmetric environment for the two iron centers for 7A (both irons 2.92) and 11A (both irons 4.60), but an asymmetric environment for 9A (Fe1 4.71, Fe2 3.11). These findings are also in agreement with our discussion in Appendix I on the spin distribution. Optical and Raman studies28 suggest that dioxygen is coordinated symmetrically to MMO; therefore, an asymmetric structure like P-cis_N (9A) should be ruled out. In light of the computed energies, structure P-cis_N (7A) should also be discarded as being too unstable. The question herein being addressed is how to proceed from intermediate P to intermediate Q. Two features characterize this conversion: (i) from an electronic point of view, a two-electron reduction (either concerted, path A in Scheme 3, or stepwise, path B in Scheme 3) is required in order to turn [FeIII(O22-)FeIII], P, into [FeIV(O2-)2FeIV], Q, and (ii) from a structural point of view, the Fe2O2 core has to be rearranged from a cis-µ-1,2peroxo form, P, to a bis-µ-oxo structure, Q. Keeping in mind a homolytic O2 activation, we have investigated two possible paths for this conversion: paths A and B in Scheme 3. The first explored path, path A, is the same for both the N- and the O-side. It would involve a two-electron

J. Phys. Chem. B, Vol. 105, No. 36, 2001 8621 transfer from the irons to the oxygens, and simultaneous O-O activation through a very symmetric trans-µ-1,2 intermediate (Scheme 3). This intermediate (P-trans in Figure 3) possesses a planar Fe2O2 diamond core with a remarkably activated O-O bond (O-O distance ) 1.664 Å). This µ-η2:η2-O2 core structure has been crystallographically well established for dicopper complexes (particularly in the structures of oxyhemocyanin and synthetic analogues29), but so far it has never been reported for diiron species. Notice that the two iron centers in P-trans would still have an oxidation state III each with approximately five spins per iron (Fe1 4.77 and Fe2 4.77 in Table 2). This would indicate that in the following step, i.e., from P-trans to Q, two electrons should be transferred from iron to oxygen simultaneously (two-electron transfer). Compound P-trans, however, has to be discarded from the operative mechanism of MMO because it is calculated to be 40 kcal/mol higher in energy than intermediate P-cis_N (11A). This result is consistent with the available experimental data mentioned above. In contrast to dicopper systems, diiron peroxo complexes with a µ-η2:η2-O2 core structure are not known. The second possible pathway, path B in Scheme 3, is a multistep path. We studied the mechanism for this path both for the N- and the O-side. Let us start describing in detail the mechanism for the N-side. The corresponding PES for the N-side of path B is shown in Figure 4. To make place for the O2 molecule, it has been suggested that one of the carboxylate ligands has to be displaced.30 Starting with peroxo intermediate P, the first step is likely to be the opening of the carboxylate leg modeling Glu 243 (see Scheme S1 for biochemical notation and details) and formation of an open intermediate, PQ0, with a dangling oxygen. With the model used in this paper, (H2O)(NH2)Fe(cis-µ-1,2-O-O)(η2-HCOO)2Fe(NH2)(H2O), such an “open” intermediate does not exist. In gas phase, the dangling oxygen is unsaturated and therefore the proposed intermediate is not stable; this oxygen either reverses back to bind Fe again (intermediate P) or tends to form an “artificial” H-bond with one of the ligands modeling either histidines or glutamates (NH2 and OH2, respectively). It should be noted, however, that a small improvement of the model (for example, by adding a water molecule on the Fe on the left) is enough to stabilize the open leg of this carboxylate. Further details on the stabilization of this intermediate due to the addition of a water ligand (and a newly proposed “water-assisted” mechanism) can be found elsewhere.31 The first intermediate following P in the present mechanism is actually PQ1. The optimized geometries for this intermediate, both in the 9A and 11A electronic states, are depicted in Figure 3. A similar structure, only for the 11A electronic state, has been reported in a previous study using a larger model.30 In the present study several isomers have been located for each electronic state. The most stable isomer (labeled PQ1_N) has a H-bond between the open carboxylate leg and the neighboring H2O ligand with a strong O‚‚‚H-O interaction. The second most stable isomer (labeled PQ1a_N) has an O‚‚‚H-N interaction. The least stable isomer (PQ1b_N) does not have any H-bond. Regardless of the existence and/or nature of the H-bond, it should be noted that the O-O bond is partially activated to similar extent in all six isomers (three 9A and three 11A) found in Figure 3. In all of them the O-O distance is within the range 1.534-1.551 Å, about 0.06-0.09 Å longer than that in P-cis isomers. Also, the O22- fragment has moved systematically from a cis disposition in P-cis to a pseudo-trans disposition in all PQ1. The spin densities in Table 2 reveal that, like P-cis, PQ1 is still formally an FeIII-FeIII species; i.e., there are no changes


Torrent et al.

Figure 3. Optimized geometries for the intermediates involved in the mechanism of the water-free conversion P f Q with O2 coming from the N-side. Bond distances are in angstroms and torsional angles in degrees.



Figure 4. Potential energy surface for the water-free conversion P f Q with O2 coming from the N-side. The energy of the reactants is taken as reference. Energies are in kcal/mol.

TABLE 2: Mulliken Atomic Spin Populations (in e) for All the Stationary Points Involved in the Reaction from P to Intermediate Q with O2 Coming from the N-side LnFe1

Fe2Ln

O2 7A

O1

O2

1.00

1.00

oxidation stateb

P-cis_N_7

2.92

2.92

0.08

0.08

FeIII FeIII (LS,LS)

P-cis_N_9 PQ0_N_9a PQ1_N_9 PQ1a_N_9 PQ1b_N_9 PQ2_9 Q_9 11A P-cis_N_11 P-trans_N_11 PQ0_N_11a PQ1_N_11 PQ1a_N_11 PQ1b_N_11 PQ2_11 Q_11

4.71 4.73 4.60 4.53 4.65 3.65 3.68

3.11 3.16 2.88 2.92 2.94 3.79 3.56

0.18 0.11 0.31 0.34 0.28 0.43 0.45

0.10 0.06 0.21 0.21 0.13 0.13 0.31

FeIII FeIII (HS,LS) FeIII FeIII (HS,LS) FeIII FeIII (HS,LS) FeIII FeIII (HS,LS) FeIII FeIII (HS,LS) FeIV FeIV FeIV FeIV

4.60 4.77 4.55 4.57 4.58 4.57 3.97 3.73

4.60 4.77 4.70 4.83 4.85 4.82 5.10 4.81

0.40 0.33 0.42 0.34 0.31 0.34 0.50 1.04

0.40 0.13 0.33 0.26 0.26 0.27 0.43 0.44

FeIII FeIII (HS,HS) FeIII FeIII (HS,HS) FeIII FeIII (HS,HS) FeIII FeIII (HS,HS) FeIII FeIII (HS,HS) FeIII FeIII (HS,HS) FeIV FeIII FeIV FeIII

9A

a Species PQ0_N_9 and PQ0_N_11 do not exist in the water-free mechanism. Spin analysis has been taken from single-point calculations on the structures obtained by removing the aqueous ligand from the optimized geometries of the analogous species (9 and 11, respectively) having an extra water molecule on Fe1. b HS ) high spin, LS ) low spin. Labels in parentheses correspond to the first and second iron, respectively.

in the oxidation state of irons from P-cis to PQ1. However, the spin density on the Fe atoms, especially on Fe1 is reduced substantially from 5.0, suggesting that a substantial “oxidation” has taken place internally, accompanying the geometrical changes discussed above. Once intermediate PQ1 is formed, the two electronic states analyzed in Figure 4 (9A and 11A) undergo clearly distinct processes. From this point of the reaction on, the two multiplici-

ties behave remarkably differently. In the 9A PES, a two-electron reduction takes place, yielding a high-valent FeIV-FeIV intermediate, PQ2 (9A). The spin densities in Table 2 confirm that the two iron atoms in PQ2 (9A) have approximately four unpaired electrons each and therefore are FeIV. Such an intermediate can easily undergo closing of the carboxylate leg to form intermediate Q (9A) without any further change in the oxidation states of the irons (because they are already FeIV in PQ2). In contrast, in the 11A PES, only one electron is transferred from Fe to O, yielding an FeIII-FeIV mixed-valence species, PQ2 (11A). As seen from Table 2, Fe2 in PQ2 (11A) has approximately five unpaired electrons (5.10) and therefore is still a d5 FeIII. However, the calculated spin density on Fe1 (3.97) reveals that this is clearly oxidized to d4 FeIV. The existence of such an FeIII-FeIV mixed-valence species in the activation of O2 by MMO, if confirmed by forthcoming experiments, would be very interesting because of its resemblance with intermediate X in ribonucleotide reductase (hereafter abbreviated as R2).32 Such an intermediate, X, has been reported to be an FeIII-FeIV species also derived from a diferric form by an intramolecular one-electron reduction.32 Moreover, our PQ2 intermediate is also in excellent agreement with recent experiments of the so-called intermediate Qx,33 an analogue of intermediate X in R2 having a mixed-valence FeIII-FeIV state. The identification of this X-like intermediate strengthens the link between the O2 reaction chemistry of MMOH and R2. It also paves the way for future studies using isotopically labeled dioxygen for the structural characterization of Q. Following the path in Figure 4, once intermediate PQ2 (11A) is formed there are two final possibilities. This intermediate can either (1) convert directly to FeIII-FeIV Q (11A) (which then has to turn into its ground-state Q (9A)) or (2) overcome spin crossing and yield FeIV-FeIV Q (9A) upon a second one-electron reduction. The results shown in Figure 4 tend to favor the latter possibility; it follows from our PES that the spin crossing should be less-energy demanding than formation of intermediate Q


Torrent et al.

Figure 5. Optimized geometries for the intermediates involved in the mechanism of the water-free conversion P f Q with O2 coming from the O-side. Bond distances are in angstroms.

(11A). Given the calculated energy differences between 9A and 11A for intermediate P (8.0 kcal/mol) and especially for intermediate PQ1 (10-20 kcal/mol), the reaction is likely to take place in the 11A-PES all along the pathway from P to PQ2. In other words, the 9A-PES does not interleave with the 11APES until the immediate predecessor of intermediate Q. Consequently, the required spin crossing of the two surfaces (from 11A to 9A) has to occur somewhere between intermediate PQ2 and Q, as marked by the small circle in Figure 4, not before PQ2. In the products Q, the relative energies for the 9A and 11A states are opposite to those in intermediate P, as seen in Figure 4. Intermediate Q (9A), an FeIV-FeIV species, is more stable than Q (11A), a mixed valence FeII--FeIV species, by 6.0 kcal/ mol. This is consistent with experimental indications34 showing that complex Q is likely to be in the FeIV-FeIV state, with four unpaired electrons per iron. The optimized geometry of species Q (9A), Figure 3, is also in excellent agreement with the experimentally reported structure of compound Q. In particular, the so-called “diamond core” Fe2O2 in the optimized structure reproduces very well the asymmetric structure found in experiment,34 with one of the diamond core O atoms closer to one Fe center, and the second one closer to the other Fe center. Both

structure and spin populations of Q (9A) and Q (11A) have been already compared and discussed in our previous paper,12 and will not be discussed in further detail here. O-Side. Starting with the active form of the enzyme MMOHred, the N-side mechanism described above is only one possibility for the O2 to bind the active site and be activated. The other possibility, more favored by experiments (Scheme 2), is to assume that O2 binding occurs on the O-side. The geometries of the intermediates involved in the O-side pathway for O2 activation are shown in Figures 1 (P-cis) and 6. The energy profile is depicted in Figure 6. In Table 3 we present the calculated Mulliken spin populations of LnFe1, Fe2Ln, O1, and O2 fragments in the optimized structures. Intermediate P-cis has the structure with the O-O moiety approaching from the H2O-side. This intermediate has already been discussed in detail in section A. As already found for the N-side, the so-called intermediates PQ0_O, 9A and 11A, do not exist in this water-free mechanism. The first actual intermediate found after P-cis in Figure 1 (or “P-cis_O” under this section) is intermediate PQ1_O. Only the more relevant/stable isomers for PQ1_O are presented here (Figure 5). These intermediates are similar to the ones already discussed previously for the N-side mechanism and will not be



Figure 6. Potential energy surface for the water-free conversion P f Q with O2 coming from the O-side. The energy of the reactants is taken as reference. Energies are in kcal/mol.

TABLE 3: Mulliken Atomic Spin Populations (in e) for All the Remaining Stationary Points Involved in the Reaction from Reactants to Intermediate Q with O2 Coming from the O-Side (See Also Table 1) structure

LnFe1

Fe2Ln

O1

O2

oxidation stateb

PQ0_O_9a PQ1_O_9 PQ2_9 Q_9 P-end-on_9 11A PQ0_O_11a PQ1_O_11 PQ1a_O_11 PQ2_11 Q_11 P-end-on_11

3.10 3.06 3.65 3.68 4.89

4.79 4.87 3.79 3.56 3.02

0.05 0.02 0.43 0.45 0.08

0.06 0.05 0.13 0.31 0.01

FeIII FeIII (LS,HS) FeIII FeIII (LS,HS) FeIV FeIV FeIV FeIV FeIII FeIII (HS,LS)

4.68 4.58 4.59 3.97 3.73 4.88

4.71 4.83 4.85 5.10 4.81 4.88

0.30 0.36 0.36 0.50 1.04 0.19

0.31 0.23 0.20 0.43 0.44 0.05

FeIII FeIII (HS,HS) FeIII FeIII (HS,HS) FeIII FeIII (HS,HS) FeIV FeIII FeIV FeIII FeIII FeIII (HS,HS)

9A

a Species PQ0_O_9 and PQ0_O_11 do not exist in the water-free mechanism. Spin analysis has been taken from single-point calculations on the structures obtained by removing the aqueous ligand from the optimized geometries of the analogous species (9 and 11, respectively) having an extra water molecule on Fe1. b HS ) high spin, LS ) low spin. Labels in parentheses correspond to the first and second iron, respectively.

discussed in detail. Only one minor aspect is worth mentioning. It should be noted that the O-O distances in PQ1_O (1.524 Å for 9A, 1.537 Å for 11A) are only 0.026 Å shorter and 0.003 Å longer than those in PQ1_N for the 9A and 11A electronic states, respectively. Comparison of Figures 3 and 6 reveals that PQ1_O (9A) and PQ1_O (11A) resemble each other more than PQ1_N (9A) and PQ1_N (11A). This is also reflected in the corresponding PESs (Figures 4 and 6, respectively). The energy difference between 9A and 11A in PQ1_O (Figure 6) is only 3.4 kcal/mol, to be compared with the 10-20 kcal/mol found in the N-side. This could be partially ascribed to the influence of the ligands trans to each oxygen atom in the O-O moiety, which is different depending on the side being analyzed, and could have a different

effect on each pair of multiplicities. Despite this reduction in the gap, the 11A PES does still lie below the 9A PES, in good agreement with our discussion for an [FeIII(O22-)FeIII] species in Appendix I. Once intermediate PQ1_O (9A) is formed, the reaction would proceed as already described in the N-side. An intramolecular two-electron reduction would yield first PQ2 (9A), which in turn would convert into Q (9A) later on. From PQ1_O (11A), there are again two possibilities as shown in Figure 6: (1) the intermediate PQ1_O (11A) could undergo a two-electron reduction and simultaneously spin-cross to the 9A PES to yield intermediate PQ2 (9A), a high-valent FeIV-FeIV; this species is ready to turn easily into Q (9A) without any change of the oxidation states of irons, and (2) alternatively, the two electrons could be transferred from Fe to O sequentially rather than in a single step; in the first step one electron would be transferred to yield the mixed-valence species PQ2 (11A), and then the second electron would be transferred near Q (9A) while crossover occurs. It is not straightforward whether the reaction proceeds through a two-electron transfer, the first possibility, or in two sequential single-electron transfers, the second possibility. According to our results in Figure 6, the most favorable pathway leading to intermediate Q (9A) seems to be the second path. In light of the results from the N-side (Figure 4) and also in light of the uncertainty of the 9A PES prior to PQ2 (Figure 6), we would favor here a sequential path over a simultaneous two-electron reduction. This path would involve a mixed-valence species, PQ2 (11A), and a spin crossing right before formation of intermediate Q. C. Comparison of N-side and O-side Pathways. Both N-side and O-side mechanisms are found to proceed through an FeIII-FeIV mixed valence species before spin crossing takes place. The existence of such a species is interesting because of the resemblance with intermediate X in R2.32 In the N-side mechanism, however, intermediate Q is not the most stable


Torrent et al.

Figure 7. Potential energy surface for the full mechanism of O2 coordination + homolytic activation, from reactants to intermediate Q, with O2 coming from the O-side. The energy of the reactants is taken as reference. Energies are in kcal/mol.

computed intermediate. This is in disagreement with experiments, which, so far, have not been able to detect any intermediate prior to Q other than P. In the O-side mechanism, the two PES’s (9A and 11A) are closer to each other than in the N-side mechanism, suggesting that the spin crossing could occur even earlier than in the N-side, i.e., nearly at any point along the reaction path. Unlike the N-side mechanism, the calculated energy profile for the O-side mechanism goes noticeably downhill from P to Q, and there are no intermediates clearly more stable than Q. This is in better agreement with experiments than the N-side approach, because (1) it has been reported that intermediate Q can be “accumulated” and (2) the lifetime of intermediate P is thought to be much shorter than Q. It should be noted, however, that the intermediates found in the O-side pathway are higher in energy than their counterparts in the N-side pathway. This fact indicates that, at least theoretically, the N-side provides a lower energy path than the O-side, which is an interesting finding. Should the protein backbone not block the N-side (via a H-bond network between His147 and Asp242, and another H-bond between His246 and Asp143, see Scheme S1, side view), the O2 molecule would most likely approach the dinuclear iron center from this side rather than from the O-side. Although our theoretical finding has little implications for the actual enzyme, where there is no other choice but binding O2 and alkanes from the hydrophobic pocket of the metalloprotein, it does have interesting consequences for the design of biomimetic compounds. The conformation of the latter can be tailored without the constraints imposed by the protein wall in the actual enzyme, and therefore, there are more possibilities than in the actual enzyme. In light of our results, bidentate ligands should be used only to mimic the residues in the O-side, but not the residues in the N-side. It is possible to select bulky ligands for the O-side without decreasing the reactivity of the biomimetic complex because the binding is likely to take place from the N-side. Moreover, there are two advantages of selecting bulky ligands for the O-side. First these ligands will provide a desirable hydrophobic cavity in the vicinity of the dimetallic

center opposite to the side where the cavity is in the actual enzyme. Second they also will serve effectively to shield against undesired bimolecular decomposition pathways which typically occur in reactions of dioxygen with diiron(II) complexes.35 On the other hand, the synthetic N-ligands employed to mimic the residues in the N-side should be small and monodentate, so that the attack of O2 from this side would not be blocked. D. From Reactants to Intermediate Q: The Full O-Side Pathway. As discussed so far, in the actual enzyme only the O-side is feasible for O2 binding. The N-side has implications only for biomimetic compounds. To better understand the operative mechanism in the real enzyme, in this section we present the full mechanism of homolytic O-O bond cleavage for the O-side, i.e., O2 coordination + activation. The full potential energy surface, from reactants to Q, is shown in Figure 7. (It should be noted that the extender, _O or _N, is not needed in this section because here we only discuss one side and therefore has been omitted everywhere below, including Figure 7). The reaction starts with coordination of the O2 molecule to the Fe-centers of the reactant, leading to formation of a mixedvalence species [FeII(O2)-FeIII], P*, with end-on coordinated terminal O2-moiety. The ground state of this complex is an 11A state. As pointed out, the O2-moiety in complex P* corresponds to a superoxo fragment, O2-, where the O-O bond has been partially activated upon one-electron transfer from the Fe atom on the right. A second one-electron transfer leads to complex P-cis, with an [FeIII(O2)2-FeIII] core and a peroxo, (O2)2-, moiety. From complex P-cis there are two possible pathways, as seen in Figure 7. Complex P-cis can convert either to complex P-endon, where the O2-moiety is coordinated to Fe-centers precisely as an end-on µ-1,1 ligand (path C in Scheme 3), or to complex PQ1, where the O2-moiety is coordinated to Fe-centers as a slightly distorted trans-µ-1,2 ligand (path B in Scheme 3). A third path, path A in Scheme 3, has been already ruled out in section B due to the instability of intermediate P-trans. Both P-end-on and PQ1 are [FeIII(O2)2-FeIII]-type diferric complexes.

Reaction of Dioxygen with Non-Heme Iron Complexes Our calculations indicate that the barriers separating P-cis from P-end-on and PQ1 should be extremely small. The barrier separating P-cis from PQ1 corresponds to raising one of the “legs” of the bidentately bridged carboxylate modeling Glu243. From complex P-end-on, the reaction is likely to proceed via the heterolytic O-O activation mechanism. Details on the heterolytic mechanism will be presented in a different paper.36 From complex PQ1, where one of the bridged carboxylates lost its bridged character and coordinated only to one of Fe centers (Fe2), the reaction proceeds via the homolytic O-O dissociation mechanism and leads to the formation of complex [FeIII(O)23-FeIV], PQ2, with a ground 11A state. (The transition state corresponding to this process is under investigation and will be reported separately). Later, the rearrangement of the carboxylate from terminal to the bridging position accompanied by subsequent electron transfer (fourth electron transfer) from the other Fe center (Fe atom on the left) leads to formation the final product, intermediate Q, [FeIV(O2-)2FeIV], with a ground 9A state. It should be noted that complex Q in the ground 9A state lies only 1.3 kcal/ mol above the ground 11A state of complex PQ2, and can be easily reached. Thus, homolytic O-O bond activation by a model complex of MMOHred includes the following steps: (i) coordination of O2 to Fe-centers to form mixed-valence complex P* accompanied by the first electron transfer to dioxygen; (ii) the second electron transfer from ferrous iron center to O2-moiety to form the cis-µ-1,2 type P complex, P-cis; (iii) raising one leg of the bridging carboxylate ligand (Glu243) and subsequent rearrangement of the O-O moiety from a cis-µ-1,2 binding mode to a distorted trans-µ-1,2 binding mode in intermediate PQ1; (iv) the activation of the O-O bond accompanied by thirdelectron transfer from Fe to O leading to intermediate PQ2, with an FeIII-FeIV mixed valence core; and finally (v) rearrangement of the carboxylate from terminal to the bridging position accompanied by the fourth electron transfer and a spin crossing, leading to the final product FeIV-FeIV, Q. IV. Conclusions From the above presented results and discussions, the main conclusions of this work can be drawn as follows. 1. Mixed-valence intermediates are not uncommon in the dioxygen activation by MMO. We have found (1) an FeII-FeIII mixed-valence species in the initial steps of the reaction (O2 coordination), and (2) an FeIII-FeIV mixed-valence species prior to formation of intermediate Q (O2 activation); the latter species is similar to intermediate X in the analogous mechanism of R2.32 Our theoretical findings give further support to the idea that electrons do not need to be transferred by pairs in the studied diiron system. Intramolecular one-electron transfers emerge as a common pattern for this reaction. 2. The energy profile for the reaction of O2 coming from the O-side is more consistent with available experimental data than for the N-side. Both pathways proceed via essentially the same intermediates, although the N-side mechanism occurs with a lower activation barrier and is also thermodynamically more favorable. In other words, if the protein backbone did not block the N-side, the O2 molecule would most likely approach the dinuclear iron center from this side rather than from the O-side. 3. Although the N-side mechanism is not feasible in the actual metalloenzyme due to the steric hindrance in that region of the protein, the fact that the N-side is more reactive than the O-side is significant for the synthesis of future biomimetic compounds. The synthetic N-ligands employed to mimic the residues in the

J. Phys. Chem. B, Vol. 105, No. 36, 2001 8627 N-side should be small and monodentate, so that the attack of O2 from this side would not be blocked. 4. The full homolytic O-O bond activation from reactants to intermediate Q includes the following steps: (i) Coordination of O2 to the Fe-centers to form a mixed-valence complex, P*, accompanied by the first electron transfer to dioxygen; (ii) a second electron transfer from a ferrous iron center to the O2moiety to form the cis-µ-1,2 type P complex, P-cis; (iii) uprising of one leg of the bridging carboxylate ligand (Glu243) and subsequent rearrangement of the O-O moiety from a cis-µ-1,2 binding mode to a distorted trans-µ-1,2 binding mode in intermediate PQ1; (iv) activation of the O-O bond accompanied by a third-electron transfer from Fe to O leading to intermediate PQ2, with an FeIII-FeIV mixed valence core; and finally (v) rearrangement of the carboxylate from terminal to bridging position accompanied by the fourth electron transfer and a spin crossing, leading to the final product FeIV-FeIV, Q.

[FeIIFeII] f [FeII(O2)-FeIII], P* f [FeIII(cis-O2)2-FeIII],P-cis f [FeIII(trans-O2)2-FeIII], PQ1 f [FeIII(O)23-FeIV], PQ2 f [FeIV(O2-)2FeIV], Q. Synthesis of a catalyst capable of dioxygen-coupled alkane oxidation based on the present results is still expected to face significant challenges. Several important features of the enzymecatalyzed reaction will be difficult to incorporate into smallmolecule mimics. For example, the other two proteins in MMO besides MMOH (reductase, MMOR and the coupling protein, MMOB) may control access of substrate to the active site, regulate electron transfer by altering the redox potential of the diiron center, and/or affect the reactivity of the various intermediates with substrates. Structural characterization of an MMOH-MMOB complex should provide considerable insight into these issues, but it is definitely far beyond the scope of the present models. Also, specific amino acids not taken into account in the investigated models may play a key role. Site-directed mutagenesis studies on cytochrome P450, for example, have revealed the importance of active site amino acid residues. In MMO, an active site cysteine and threonine not directly bound to the iron centers may have other important mechanistic consequences. Although the significance of these residues is not yet known, it may be necessary to consider their roles when designing synthetic catalysts. Further model studies including the backbone of the protein are currently being conducted in our laboratory. Acknowledgment. The present research is in part supported by a grant (CHE96-27775) from the National Science Foundation. M.T. gratefully acknowledges a Postdoctoral Fellowship from the Spanish Ministerio de Educacio´ n y Cultura. Acknowledgment is made to the Cherry L. Emerson Center of Emory University for the use of its resources, which is in part supported by a National Science Foundation grant (CHE-0079627) and an IBM Shared University Research Award. Computer time allocated at the Center for Supercomputing Applications (NCSA) and Maui High Performance Computer Center (MHPCC) is also acknowledged. Supporting Information Available: Cartesian coordinates and 〈S2〉 values of all 31 stationary points, one scheme showing front view and side view of the dinuclear active site including several neighboring amino acids (Scheme S1), and Appendix I

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