Phenol-Induced O–O Bond Cleavage in a Low-Spin Heme–Peroxo–Copper Complex: Implications for O2 Reduction in Heme–Copper Oxidases

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Video Phenol-Induced O–O Bond Cleavage in a Low-Spin Heme–Peroxo–Copper Complex: Implications for O2 Reduction in Heme–Copper Oxidases

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3.1. Reaction Coordinate of 1 with Exogenous Phenol

In order to investigate the observed reactivity of 1 toward exogenous phenolic substrates, a model containing both reactants was constructed (1·PhOH) using an unsubstituted phenol. The geometric and electronic structure of 1 has been defined previously (using the BP86 functional, shown to provide good agreement with EXAFS and resonance Raman data),33 and is employed as a starting point for this study. The broken symmetry MS = 0 state, achieved by AF coupling between the low-spin FeIII and CuII ions, is lowest in energy by 1.7 kcal/mol (ΔG) (consistent with experiment). The starting point for surveying this reaction was obtained from an unconstrained optimization of 1 with an approaching PhOH, which yielded an energetic local minimum denoted the docked reactant, D (), that is 7.0 kcal/mol more stable in ΔE than the separated species (3.3 kcal/mol less stable in ΔG, owing to the entropic cost of bringing the components together).41 In the docked reactant structure, the phenol is H-bonded to the peroxo O on the Cu (OCu) with an OCu⋯O(H)Ph separation of ∼2.8 Å.42 The H-bonding interaction induces a slight elongation of the peroxo O–O bond from 1.40 Å (in 1) to 1.43 Å. The low-spin Fe(III) has a doubly-occupied dyz orbital (in the Fe–O–O plane) and a singly occupied dxz orbital (perpendicular to the Fe–O–O plane), with the latter having π overlap with the peroxo π* orbital (by convention the Fe is designated as having α-spin). Note that the dxz and dyz orbitals on Fe have π overlap with the O22−π* and σ* orbitals, respectively (Figure S1).

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(A) Starting structure (D) for 1·PhOH, having O⋯O, OCu⋯ H(OPh), and OCu⋯OPh(H) distances of 1.43, 1.75, and 2.73 Å, respectively. (B) Product structure (P) for 1·PhOH, with distances of 3.54, 0.99, and 2.79 Å, respectively. H-atoms have been removed for clarity (except the phenolic H).

Given that the experimentally observed products include a phenoxyl radical (vide infra), transfer of a proton and an electron from the phenol to 1, followed by an unconstrained optimization yielded a product structure, P, consisting of three fragments: [(F8)(DCHIm)FeIV=O], [(AN)CuII(OH)]+, and the associated phenoxyl radical (). On the MS = 0 surface, these fragments have spins of MS = 1, MS = −1/2, and MS = −1/2, respectively. Thus, over the course of the reaction, a β electron has transferred from the Fe dyz orbital, and an α electron (and H+) has transferred from the PhOH. This phenoxy-associated product has an energy ΔG = −12.0 kcal/mol relative to the docked reactants (ΔE = −6.1 kcal/mol). Separating the phenoxyl radical yields the overall thermodynamics for the separated reactants to separated products of ΔG = −7.9 kcal/mol (ΔE = −5.3 kcal/mol), comparable to the values obtained for the associated species.

3.1.1. The Overall Reaction Landscape

The PES connecting the reactant and product minima described above was calculated as a function of three coordinates: the OFe—OCu, OCu⋯H(OPh), and OCu⋯OPh distances. Two representative surface slices with the OCu⋯OPh coordinate fixed at 2.4 and 2.6 Å (which are close to the OCu⋯OPh distance for two key saddle points identified on the 3D surface, vide infra) are shown in , respectively. Examination of the combined potential surface revealed two possible reaction pathways, approximated by the red and blue curves in . As can be seen from the figure, one pathway (red curve) involves an initial, nearly complete decrease in the OCu⋯H distance, followed by OFe–OCu elongation, therefore indicating that the H+ transfer to OCu occurs early in O–O cleavage. The second pathway (blue curve) involves nearly complete OFe–OCu elongation followed by the decrease in OCu⋯H, indicating that the H+ transfer occurs late in O–O cleavage. With respect to the current literature on CcO,19,27,43–46 the mechanism depicted by the first pathway is closely representative of the process that has generally been considered in the enzyme. To evaluate which mechanism is kinetically favored in {1+PhOH} (i.e., which has the lowest barrier), a TS was found for each pathway.

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PESs for OFe⋯OCu vs OCu⋯H(OPh) distances (with fixed OCu⋯OPh), where the top/bottom plots are rotated ∼90° from each other to show the full surface. (A) OCu⋯OPh = 2.4 Å; the red line represents an approximate reaction pathway through the proton-initiated TS (TSPI). (B) OCu⋯OPh = 2.6 Å; the blue line represents an approximate reaction pathway through the H-bonded TS (TSHB). All energies are relative to the structure with OCu⋯OPh = 2.6 Å, OFe–OCu = 1.4 Å, and OCu⋯H(OPh) = 1.6 Å (which is set to 0 kcal/mol). Dashed lines are used to indicate that the path is behind the surface.

Using the highest energy point along the first reaction pathway (red curve) as an initial guess, an unconstrained TS was found (shown in ) with an energy of ΔGǂ = 7.9 kcal/mol above the docked structure (ΔEǂ = +11.7 kcal/mol, which neglects zero-point corrections to the total energy that lower the barrier by ∼3.5 kcal/mol). This TS structure has the H+ almost completely transferred to the OCu (the OCu⋯H distance is 1.12 Å, relative to 0.99 Å in the Cu–OH product shown in ), yet is relatively early in the O–O coordinate with a bond length of 1.88 Å, compared to 1.43 Å in D (). Note that the OCu⋯OPh separation is 2.45 Å, significantly shorter than in D (2.73 Å). The imaginary frequency in the TS is predominantly H motion between OCu and OPh, and to a lesser extent, O–O elongation and phenolate rotation. Importantly, the phenolic electron still resides on the phenolate ring at the TS (from a population analysis, vide infra), indicating only the proton is transferred, rather than an H-atom. Given the nature of the transition state, this mechanism for O–O cleavage is referred to as “proton-initiated” (PI), and the transition state is denoted TSPI.

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Geometric structures for (A) the transition state along the proton transfer pathway (TSPI), and (B) the transition state along the H-bond-assisted O–O homolysis pathway (TSHB). Green arrows illustrate the dominant motion in the imaginary frequency. Bond distances are given in the SI, Table S1. H-atoms have been removed for clarity (except the phenolic H).

In a similar manner, a TS for the second reaction pathway (blue curve in ) was found having an energy of ΔGǂ = +10.0 kcal/mol above the docked reactant (ΔEǂ = +10.9 kcal/mol). The structure of this TS is depicted in , where the H is still predominantly on the phenol (the H–OPh bond length has increased to 1.04 Å, from 1.00 Å in the docked reactant), although the OCu⋯H distance has decreased from 1.75 to 1.54 Å due to the phenol moving closer to the peroxo core (the OCu⋯OPh distance has decreased from 2.73 Å in D to 2.57 Å in the TS). Thus, in this TS the proton has only minimally transferred and the phenol is effectively serving as an H-bond donor as the O–O bond is homolytically cleaved. The imaginary mode in this TS primarily involves O–O elongation, while the phenol moves in concert with OCu, such that the OCu–H distance exhibits very little change. Given the nature of the transition state, this reaction pathway is referred to as “H-bond assisted O–O homolysis” (or “HB”), and the transition state is denoted TSHB.

Comparing the reaction barriers calculated for these two pathways, the PI reaction coordinate is predicted to be kinetically favored, as it is lower in ΔGǂ by 2.1 kcal/mol. However, given that they are fairly similar in energy, and have sufficiently low barriers to be kinetically feasible, we examined each reaction coordinate to understand how and why they differ, and to confirm that both lead to the correct products.

3.1.2. Proton-Initiated O–O Cleavage (PI Mechanism)

The PES for the PI reaction pathway () was generated by following the intrinsic reaction coordinate (IRC) from TSPI in the reverse and forward directions, which yielded D and P, respectively. The reaction coordinate up to the barrier (from D to TSPI, ) involves three key geometric changes: the OFe–OCu bond lengthens, the Fe–O bond shortens, and the OCu⋯H distance shortens. These changes in bonding are coupled to electron transfer from Fe into the peroxo (predominantly β-spin, yielding an S = 1 ferryl heme) (Figure S3), which derives from the Fe dyz orbital via π–backbonding into the peroxo σ* orbital (Fe dπ(σ*) in and Figure S2). The O–O bond elongation from 1.43 to 1.88 Å parallels the increase in occupation of the σ* orbital () and decrease in O–O Mayer bond order (MBO) (from 0.85 to 0.43, ), suggesting that the bond is approximately halfway cleaved of TSHB. Meanwhile, the Fe–O bond shortens from 1.82 to 1.68 Å and the respective MBO increases from 0.68 to 1.11, indicating significant Fe–O double bond character at the TS. Finally, the OCu–H distance shortens from 1.75 to 1.12 Å and the MBO increases from 0.12 to 0.45. This is assisted by the electron density transferred from Fe, which localizes primarily on OCu (Figure S3A,B), thereby increasing the negative charge on OCu and strengthening its interaction with the approaching H(OPh).

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PES vs OFe–OCu distance for the PI pathway, generated by an intrinsic reaction coordinate from the TS (TSPI), with the OCu⋯H and OCu⋯OPh vectors unconstrained. The structure at TSPI is shown in and described in the text.

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(A) Mayer bond orders for Fe–O, O–O, Cu–O, OCu–H, and OPh–H. Population densities for (B) α-spin and (C) β-spin Fe dπ orbitals, O–O σ*, and PhO HOMO correlated to O–O distance over the IRC. The Fe dπ orbitals are differentiated by their overlap with the peroxo MOs. O–O σ* orbital occupancy is only reported up to ∼40% occupation. The vertical dashed lines mark the O–O distance in TSPI.

A key characteristic of the TSPI electronic structure is that both α and β HOMOs of phenolate (331α and 331β in Figure S2) are fully occupied, indicating that only the proton, and not the electron, has transferred from the phenol at the TS (Figure S2). Additionally, the charge on the porphyrin ring increases (becomes less negative) prior to the TS, resulting from polarization of the Fe–N σ bonds toward Fe (see Figure S5 and text) to compensate charge donated out of Fe. Taken together, these results establish that Fe is the primary source of e transfer into the σ*O–O up to the TS. The overall electronic structure of TSPI is therefore best described as FeIV=(O—OH)2−—CuII/PhO, where the O2 moiety is now three-electron reduced.

The PI reaction coordinate after the TS comprises two key processes: transfer of the second electron required for full reduction of O22−, and completion of the O–O bond cleavage. Since the electron transferred from Fe (which is mostly complete by the TS) has β-spin, the second electron has α-spin. From (also Figure S3B), the α e derives from phenolate, which transfers into the σ*O–O over the remainder of the reaction coordinate. This involves donation out of the phenolate HOMO, which, following a rotation of the phenol ring (∼30°, see Figure S4), has direct overlap with the σ*O–O through the out-of-plane p(π)-orbital on the phenolate oxygen (depicted in the MO contours in ). Since this completes O2 reduction, the O–O bond cleaves and the FeIV= O, CuII—OH, and PhO fragments move apart. An unconstrained optimization thus yields the products shown in , giving an overall ΔG = −12 kcal/mol.

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Occupied (bottom) and virtual (top) α-spin molecular orbitals comprised primarily of the Fe dyz, O–O σ*, and PhO HOMO, shown at increasing O–O distances of 1.88, 2.25, and 2.6 Å. The change in population density illustrates the progressive transfer of an α electron from phenolate (occupied at the TS) into the O–O σ* (which increases in occupation).

3.1.3. H-Bond-Assisted O–O Homolysis (HB Mechanism)

The second pathway for the reaction of {1+PhOH} (blue in ) involves O–O bond cleavage with minimal H+ transfer from phenol at the transition state (TSHB, ). During the reaction coordinate from D to TSHB (generated from the reverse IRC from TSHB, shown as the blue curve in ), the O–O bond elongates while the Fe–O and Cu–O bonds shorten, and the phenol moves closer to OCu but remains an H-bond donor. These changes are coupled to the transfer of a β electron from Fe into the σ*O–O orbital, which polarizes toward OCu (similar to the PI reaction pathway, see Figure S7). The O–O bond has elongated from 1.42 Å in D to 2.30 Å in TSHB (longer than the 1.88 Å bond length in TSPI), although the energy rises minimally after reaching an r(O–O) of ∼2.0 Å. Based on an O–O MBO of 0.19 in TSHB (decreased from 0.85 in D, ), the O–O bond is nearly cleaved (although the fourth e has not yet transferred). In concert, the Fe–O bond has shortened from 1.82 to 1.66 Å, and the Cu–O bond has shortened from 1.95 to 1.84 Å, with bond orders likewise reflecting much stronger bonds at the TS (). In addition, the OCu⋯H distance of 1.54 Å in TSHB indicates that the phenol is H-bonded to the peroxo moiety, though it is worth noting that this interaction strengthens as the reaction proceeds to the TS, as evidenced by the increase in OCu–H MBO from 0.12 to 0.21. The overall electronic structure of TSHB is best described as an FeIV=O/CuII—O/PhOH, where both metal fragments are triplet species (α for Fe, β for Cu). Due to the orthogonality of the singly occupied orbitals involved (Figure S8), a triplet Cu-oxyl is 3.8 kcal/mol more stable47 than the singlet species that would form on the overall triplet surface.

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PESs for O–O cleavage without phenol (purple), with phenol H-bonded (blue), and with the phenol H+ transferred to the peroxo (green) vs OFe–OCu separation.

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Mayer bond orders along the HB reaction coordinate, without H+/e transfer from phenol.

Continuing in O–O elongation past TSHB, an IRC calculation in the forward direction yields a structure having a fully cleaved (3.5 Å, far right on blue curve in ) O–O bond, an FeIV-oxo porphine, a CuII-oxyl, and a phenol still H-bonded to the oxyl. To clarify the electronic structure of this product, we note that oxidizing the porphyrin (to form a porphyrin radical and CuII-oxo) would be 43 kcal/mol higher in energy, and oxidizing the phenol would be 66 kcal/mol higher. Thus, without H+ transfer from phenol, Cu-oxyl formation is driven by thermodynamics. Nevertheless, the energy of this structure is 4.1 kcal/mol (ΔG, 7.4 in ΔE) above D, which confirms that H+ transfer from phenol is necessary for O–O cleavage to become thermodynamically favorable.

Therefore, to investigate the process of transferring the H+ and e to the Cu-oxyl to reach the favorable phenoxyl radical-containing products that are experimentally observed, an additional PES scan was generated. This involved fixing the proton initially from the phenol on the peroxide OCu in the reactant and elongating O–O bond (, green curve). Comparing this to the IRC containing TSHB (, blue curve), these surfaces become isoenergetic at r(O–O) ≈2 Å, after which it is thermodynamically favored for the H+ to transfer to OCu. However, transferring the H+ at the crossing point involves an additional barrier (∼1.5 kcal/mol) that is greater than the ∼0.5 kcal/mol required to reach TSHB (at r(O–O) = 2.3 Å), and therefore the lowest energy reaction pathway follows the H-bonding O–O homolysis surface (blue curve). As the O–O bond continues to elongate and it becomes increasingly more favorable for the H+ to reside on OCu, the H+ eventually transfers without contributing to the barrier (see SI for details and evaluation of this process). However, when the H+ can transfer, the energy (along the blue curve) has already reached that of TSHB (which is therefore ΔGǂ). Interestingly, when the H+ is halfway transferred, the phenolate still has <10% radical character (from a Mulliken analysis, Figure S17), indicating that the net H-atom transfer occurs as PT, followed by ET, similar to the “proton-initiated” pathway through TSPI described in section 3.1.2. Following H+ and e transfer, a full optimization of the resultant structure yielded the final products P in .

Given that this reaction pathway essentially represents O–O homolysis with an H-bond donor interacting with OCu, it is instructive to compare this (blue curve in ) to O–O homolysis without the phenol present (purple curve in ). From , the H-bond lowers the barrier from 17.5 to 10.0 kcal/mol in ΔGǂ (from 17.6 to 10.9 in ΔEǂ, purple and blue curves, respectively), which is due to the H-bond enhancing electron donation from Fe to promote O–O cleavage (see section 3.1.4 below).

Overall, this reaction pathway illustrates how an H-bond donor can lower the barrier to O–O homolysis by increasing donation from Fe and thereby raising the H+ affinity of the peroxo moiety, enabling fast PT-ET to form the thermodynamically favorable FeIV=O, CuII—OH, and PhO, where the reaction barrier is defined by the H-bonded O–O homolysis and not the H+/e transfer. While these calculations yield a slightly higher energy barrier relative to the “PI pathway” in which the proton transfers much earlier in O–O cleavage (10.0 vs 7.9 kcal/mol), both pathways demonstrate that the phenolic proton serves to lower the barrier to O–O cleavage, and that the PT precedes ET, which occurs after the barrier.

3.1.4. Effect of Metal–Ligand Covalency on the Reaction Surface

The above PESs, structures, and thermodynamics were obtained using the pure density functional BP86, which was shown to provide reasonable agreement with structural (EXAFS) and vibrational data at a lower computational cost than a hybrid functional (such as B3LYP). However, it is well documented that introducing the Hartree–Fock (HF)exchange in a hybrid functional will alter the bonding such that metal–ligand (M–L) interactions become less covalent.48–52 Indeed, when compared to BP86, a calculation of 1 in B3LYP yields lower Mayer bond orders for the Fe–O, Fe–N, O–O, and Cu–N interactions (Cu–O is unchanged), along with greater charges on these atoms, demonstrating the less covalent M–L bonding (see Table S2 for comparison of B3LYP and BP86). Applying B3LYP to 1·PhOH yields the same trends in the bonding of the docked reactant structure, D. We therefore examined how the inclusion of 20% HF exchange in a B3LYP calculation qualitatively and quantitatively impacts the {1+PhOH} reaction surface.

As summarized in , there is a generally good agreement in thermodynamics (ΔG°) calculated using the two functionals. We note that the difference in thermodynamics calculated for the fully optimized interacting products (“Proton-initiated O–O cleavage” in the table) is attributable to the difference in docked product structures predicted by the two functionals (see Figure S9 for details).

Table 1

Comparison of Barriers and Thermodynamics for O–O Cleavage Using BP86 and B3LYP

reactionb ΔGǂEǂ)a G° (ΔE°)a

O–O homolysis of 1 (no PhOH, yielding CuII–O) 17.5 (17.6) 20.2 (20.6) +8.8 (+9.8) +10.1 (+10.9)
H-bond-assisted O–O cleavage (without PT from PhOH, yielding CuII–O) 10.0 (10.9) 16.4 (17.5) +4.1 (+7.4) +5.6 (+8.3)
Proton-initiated O–O cleavage (yielding P) 7.9 (11.7) 24.3 (25.7) −12.0 (−6.1) −5.8 (−2.0)
{1} + {PhOH} → {FeIV=O + CuII—OH} + {PhO} −7.9 (−5.3) −9.9 (−7.4)

To systematically evaluate how the barriers and PESs are affected, we first consider the homolytic O–O cleavage without phenol present (plotted as the purple curves in for B3LYP and BP86, respectively). A comparison between the two functionals reveals that there is only a small energetic difference in the barriers, where B3LYP yields a TS that is 2.7 kcal/mol higher in ΔGǂ () and occurs at a shorter O– O distance (1.95 Å, vs 2.3 Å in BP86).

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Comparison of B3LYP and BP86 PESs for the O−O homolysis without phenol (purple), PI pathway (red), H-bond assisted O−O homolysis (blue), and the phenolic H+ transferred and optimized on the peroxo (green) vs OFe−OCu separation. Note that the TSPI in B3LYP was obtained by increasing and fixing the OCu−OPh distance to 2.6 Å to prevent the H+ from returning to the phenolate, as an unconstrained transition state search converged to the much lower energy TSHB. Changing the OCu−OPh distance was found to have only a minor effect on the energy when the phenol was deprotonated. The dotted line denotes that the relaxed PES through TSPI requires an additional structural constraint and therefore is not generated from an IRC.

We next evaluated the reaction pathway in {1+PhOH} where the phenol is H-bonded to the cleaving peroxo for the two functionals (blue curves in ). The H-bond lowers the barrier (relative to O–O homolysis) by only 3.8 kcal/mol in B3LYP, compared to 7.5 kcal/mol in BP86. Since the homolysis of 1 was already slightly higher in B3LYP, the net result is that the barrier to H-bond assisted O–O homolysis (forming the FeIV=O/CuII—O/PhOH species) is 6.4 kcal/mol higher in B3LYP than BP86, which is due to the difference in covalency (vide infra).

Finally, comparing the barrier to O–O cleavage when PT occurs prior to the homolysis barrier (red curves through TSPI in ), B3LYP predicts a much higher energy for TSPIGǂ = 24.3 kcal/mol) than TSHBGǂ = 16.5 kcal/mol), while in BP86 the TSPI barrier was lowest (2.3 kcal/mol lower than TSHB, vide supra). In fact, the TSPI in B3LYP (in which the H+ is almost completely transferred to OCu) is at sufficiently higher energy that it is unstable to the H+ returning to the phenolate (thus requiring a structural constraint, see caption). Therefore, the lower covalency not only quantitatively changes the barrier heights, but also qualitatively changes the relative barriers and therefore the predicted mechanism, by causing the PI pathway to be higher in energy in the less covalent B3LYP calculation.

Insight into how the interaction with the proton and the change in covalency each affect the barrier to O–O cleavage can be gained from an evaluation of the PESs generated by transferring the H+ to OCu in the reactant and elongating the O–O bond (green curves in ). Comparing these surfaces shows that in BP86 the barrier to cleave the peroxo O–O bond is effectively removed, while in B3LYP there is still a significant barrier. This is because the interaction with the proton (or H-bond) lowers the energy of the peroxide MOs, therefore allowing easier electron donation from Fe. Since cleaving the O–O bond requires ET from Fe into σ*O–O, this results in a lower barrier.

The more covalent M–L bonding (in BP86) also facilitates donation from Fe into σ*O–O, likewise resulting in lower barriers to O–O cleavage (compared to B3LYP). The difference in the PESs for cleaving the protonated peroxo (green curves in ), where only the B3LYP calculation shows a barrier, reflects the fact that there is initially little electron transfer from Fe into σ*O–O in B3LYP (from MBO and Mulliken analyses, see SI section 5), while in BP86 the H+ enhances electron flow and therefore accelerates O–O homolysis. Additionally, greater backbonding from Fe (due to higher covalency) increases negative charge on the peroxo, which raises the proton affinity of the OCu-atom relative to the phenolate (making H+ transfer favorable earlier in O–O cleavage).

Overall, the BP86 calculation yields two possible reaction pathways that have energetic barriers defined by TSPI and TSHB, which are similar in energy (with TSPI slightly lower) and lead to the same products. In contrast, upon altering the bonding description to be less covalent by using B3LYP, the latter barrier is significantly lower and therefore the H-bond assisted pathway is favored. The relative barrier heights depend on two competing factors: the degree to which protonation of the peroxo lowers the barrier to O–O cleavage (compared to H-bonding), and the energetic cost of deprotonating the phenol. That is to say, in BP86, protonating the peroxo lowers the barrier to O–O cleavage enough to overcome the proton affinity of the phenolate. Conversely, in B3LYP, protonation of the peroxo does not sufficiently lower the barrier, so the proton transfers later in O–O cleavage, after the O–O homolysis barrier.

3.2. Experimental Evaluation of the O–O Cleavage Mechanism

Given that two DFT functionals (BP86 and B3LYP) provided qualitatively and quantitatively different descriptions of {1+PhOH}, a computational evaluation alone cannot reliably elucidate how an exogenous phenol induces O–O bond cleavage in 1. Therefore, further experimental data were obtained to determine the mechanism by which this occurs and evaluate the reaction barrier.

To first consider which functional more accurately models the covalency of 1, we can turn to spectroscopic data. In our earlier study,33 we found that TD-DFT calculations performed using B3LYP more accurately predicted the O22− to Fe3+ charge-transfer transition and provided good agreement with the resonance Raman profile, indicating that B3LYP offers a better description of the covalency in 1. It was suggested that B3LYP provided better agreement with the data because BP86 predicted the porphyrin orbitals to be too high in energy, resulting in overly covalent Fe-porphyrin bonding.

To evaluate the possible reaction mechanisms presented in section 3.1, we investigated the reaction of 1 with an H-atom-donating phenol, 4-OMe-PhOH, which accomplishes this reaction at low temperature (−70 to −80 °C, where 1 is stable, ).53 To verify that the observed reactivity is comparable to the reaction modeled by DFT, we have demonstrated that (1) the phenoxyl radical is formed, (2) the addition of 4-OMe-PhOH accelerates the disappearance of 1, and (3) decomposed 1 is unreactive toward phenol (see SI).

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Reaction of 1 with 4-OMe-PhOH Under Substrate-Saturated Conditionsa,b

aUnder these conditions, kinetic measurements are independent of [4-OMe-PhOH]. bWe postulate that the (DCHIm)F8FeIV=O product is highly reactive and rapidly abstracts an H-atom from an additional phenol, yielding the FeIII—OH product observed experimentally.

Under substrate-saturated conditions where the rate is independent of phenol concentration (), we obtain a reaction rate of kobs = 4.18 × 10−4 s−1 at −70 °C, corresponding to a barrier of ΔGǂ = 14.9 kcal/mol. This value is very similar to the ΔGǂ (14.8 kcal/mol) obtained from an Eyring analysis of the rates measured at −70, −72, −74, and −77 °C (, inset). Accordingly, the reaction barriers for the PI and HB pathways were recalculated (at −70 °C) with 4-OMe-PhOH, yielding values for BP86 (ΔGǂPI = 7.2 kcal/mol, ΔGǂHB = 8.3 kcal/mol) and B3LYP (ΔGǂPI = 20.9 kcal/mol, ΔGǂHB = 16.2 kcal/mol) that were comparable (in both energy and TS geometry) to the unsubstituted phenol used in section 3.1. Note that these values are calculated relative to an optimized “docked” structure, as the experimental rate is saturated in [4-OMe-PhOH] (analogous to Michaelis–Menten conditions in steady-state kinetics).54 Thus, the barrier predicted by BP86 (7.2 kcal/mol) underestimates the experimental value of 14.9 kcal/mol, while the barrier in B3LYP (16.2 kcal/mol) overestimates it.

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(A) Absorption spectra during the reaction of the LS-AN complex (1) (0.1 mM, −70 °C, MeTHF) (red) with >50 equiv 4-methoxyphenol to form the F8FeIII–OH final products (blue). (A, inset) Time evolution of absorption changes corresponding to 1 (533 nm) and FeIII–OH (555 nm). (B) Plots of initial reaction rate (monitored at 555 nm) dependence on phenol concentration at −70, −72, −74, and −77 °C, including Michaelis–Menten parameters for each temperature. (B, inset) Eyring analysis of the kinetic data, using the Vmax for each temperature.

However, given that the two mechanisms differ qualitatively by the extent of proton transfer from phenol involved in reaching the TS (where one involves predominantly proton transfer, while the other involves minimal proton transfer), these can be experimentally distinguished by KIE measurements. Employing a deuterated 4-OMe-PhOD slows the reaction rate to kobs = 2.41 × 10−4 s−1 at −70 °C, yielding a KIE of kH/kD = 1.7 (). As expected from the nature of TSPI and TSHB, a larger primary KIE (kH/D = 7.7 in BP86, 10.2 in B3LYP) is calculated for the former, while a smaller secondary KIE (kH/D = 1.6 in BP86, 1.2 in B3LYP) is calculated for the latter, based on the PhOH/PhOD effect on ΔGǂ. Thus, while the barrier calculated for each mechanism varies greatly with functional, a KIE >5 is predicted for the PI mechanism, compared to a KIE <2 for the HB mechanism, independent of functional (Table S7).55Taken together, these results indicate that the reaction of 1 with 4-OMe-PhOH proceeds via the HB mechanism.

Table 2

Summary of Kinetics Data Using the Reaction Conditions Given in
{1 + p-OMe-PhOH} kinetics data (50 equiv, –70 °C)a
kobs(p-OMe-PhOH) (s−1) 4.18 (0.2) × 10−4
kobs(p-OMe-PhOD) (s−1) 2.41 (0.4) × 10−4
KIE (kH/kD) 1.7 (0.3)
ΔGǂ (kcal/mol) 14.9 (0.1)

In the interest of determining a method that accurately reproduces the experimental results, we calculated the barriers to both mechanisms for {1 + 4-OMe-PhOH} using several functionals that are commonly employed for first-row transition metal complexes in the literature and vary in the amount of HF exchange (M06-L, TPSSh, ωB97X-D, and PBE0). These results (Tables S6 and S7) further illustrate the trends observed between BP86 and B3LYP, where an increase in HF exchange yields a higher barrier and more strongly favors the HB mechanism. Based on these calculations, TPSSh and B3LYP appear to most accurately reproduce the experimental barrier, while BP86 provides the closest estimate for the KIE (for the HB mechanism).

3.3. Correlation to CcO

The above calculations and experimental data establish that the transfer of a H+/e pair from phenol enables favorable O–O bond cleavage in 1. It is therefore valuable to consider how these results relate to cytochrome c oxidase, in which a cross-linked Tyr residue (that is ∼6 Å away from CuB) is widely proposed to participate in O–O cleavage. As a benchmark comparison to {1+PhOH}, we first evaluate the reaction of an exogenous phenol with a bridging peroxo species in CcO, using a model of the active site that includes the cross-linked Tyr (). Similar to {1+PhOH}, if the exogenous phenol is allowed to provide both the H+ and e, the reaction is exergonic by 3.1 kcal/mol (calculated in a dielectric of 4.0 at room temperature), compared to –7.9 kcal/mol for 1. The key implication of this for the cross-linked Tyr residue (i.e., a separated phenol) is that it could induce thermodynamically favorable O–O cleavage in CcO by supplying both the H+ and e, analogous to the reaction of {1+PhOH}.

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Computational model of the cytochrome c oxidase active site, based on the crystal structure from bovine heart (PDB code 1V54). The α carbons of all included residues are frozen to mimic constraints imposed by the protein backbone.

However, given that the calculations in section 3.1 indicate that the net H-atom transfer from phenol in {1+PhOH} occurs in a stepwise manner as PT followed by ET, independent of the timing of H+ transfer (before or after the barrier), it is important to consider that in CcO, multiple proton and electron donors are available. This is in contrast to {1+PhOH}, where the phenol is the only possible source of the proton. Following PT from phenol to 1, the electron can derive from either the resultant phenolate or the heme fragment. Oxidation of the heme would generate an FeIV=O/porphyrin radical species (called “Compound I”) that is known to form in many porphyrin systems in biology such as cytochrome P450 enzymes and peroxidases, and has even been invoked in some computational studies on CcO.19 A straightforward explanation for why a porphyrin radical species is not observed in {1+PhOH} lies in the thermodynamics of phenolate oxidation versus porphyrin oxidation. Calculation of the ionization potentials for the individual PhO and [(DCHIm)-(F8)FeIV=O] fragments reveals that the heme is 36 kcal/mol harder to oxidize than the phenolate. Since the PhO is oriented such that its electron can easily transfer to the peroxy moiety (), the thermodynamics govern the reaction products and a Compound I intermediate would therefore not form in {1+PhOH}.

In CcO, while the cross-linked Tyr residue is generally implicated in the O–O cleavage step of the catalytic cycle, the nature of the proton donor remains unknown. However, given that in the reaction of reduced CcO with O2, no proton uptake or translocation to the active site occurs before forming PM (the first intermediate after the O–O bond is cleaved),56,57 the H+ must derive from an amino acid or other donor within the enzyme. Furthermore, since a peroxo-level intermediate is not observed in CcO during the generation of PM, the H+ donor must be near the active site for the protonation and reduction step to occur rapidly. The possible proton sources within a ∼10 Å radius of the active site include Tyr244, Thr309, Thr316, Asp364, His368, Arg438, and propionic acid pendants of heme a3.

Since the source of the fourth electron required for O2 reduction has likewise been disputed in the literature,58,59 we next considered possible electron donors around the active site. Based on thermodynamics (summarized in and ref 60), none of the available proton donors are of sufficiently low energy to drive O–O cleavage via oxidation of heme a3 (forming a porphyrin radical),61 indicating that the electron must also derive from a protein residue.62

Table 3

Overall Thermodynamics of O–O Bond Rupture Forming an FeIV=O/Por/CuII—OH Species in CcO, Employing Several Common Amino Acids and Some Small-Molecule Donors for Reference

{[(heme a3)(His)Fe–O2–Cu(His)3]+} + {HA} → {[(heme a3)FeO(His)]+ + [(His)3Cu(OH)]+} + {A}

H+ donor ({HA}) ΔG° (kcal/mol)a
Arg+ +10.3b
Asp +29.9
His+ 0b
Thr +55.2
Tyr +41.6
CH3COOH +40.6
PhOH +44.4

Examining the crystal structure, the possible electron donors include Trp126, Trp236, and the cross-linked Tyr244, all three of which have been proposed to participate in the redox chemistry of CcO.58,59 Evaluating the thermodynamics for O–O cleavage driven by protonation (from one of the possible H+ donors) and reduction by each of these two amino acids, it was found that the only e source that enables a favorable reaction is a deprotonated Tyr. As shown in , even employing the lowest energy H+ donor that is realistically possible in CcO (Arg+, from ), oxidation of Trp to cleave the O–O bond would be unfavorable by 12.7 kcal/mol (oxidation of a protonated Tyr is an additional 4.5 kcal/mol uphill).66 These results indicate that the deprotonated Tyr residue likely serves as the active reductant during the O–O cleavage step. Importantly, this in turn necessitates that the cross-linked Tyr is deprotonated at the time that the peroxo is cleaved. Furthermore, if Tyr serves as both the H+ and e donor, O–O cleavage and formation of the tyrosyl radical is favorable by 4.6 kcal/mol, which is in agreement with the ∼4 kcal/mol estimate for the driving force of this step in CcO based on studies calibrated to kinetic data on the enzyme.67 Overall, the thermodynamics indicate that the cross-linked Tyr is required to effectively act as the proton donor in order to generate a tyrosinate, the only accessible electron source that leads to favorable O–O cleavage.

Table 4

Overall Thermodynamics of O–O Bond Rupture in CcO for Different Combinations of Possible e Source with the Lowest Energy H+ Donor Available (Arg+) and Tyra

{[(heme a) (His)Fe–O2-Cu(His)3]+} + {HA} + {E} → {[(heme a) FeO(His)]+ + [(His)3Cu(OH)]+} + {A} + {E}

H+ donor ({HA}) e donor ({E}) ΔG° (kcal/mol)
Arg+ Trp +12.7
TyrH +17.2
Tyr −35.9
Tyr Trp +44.0
Tyr −4.6


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