Geometric and Electronic Structure Contributions to O-O Cleavage and the Resultant Intermediate Generated in Heme-Copper Oxidases

Abstract

This study investigates the mechanism of O-O bond cleavage in heme-copper oxidase (HCO) enzymes, combining experimental and computational insights from enzyme intermediates and synthetic models. It is determined that HCOs undergo a proton-initiated O-O cleavage mechanism where a single water molecule in the active site enables proton transfer (PT) from the cross-linked tyrosine to a peroxo ligand bridging the heme Fe^III and Cu^II, and multiple H-bonding interactions lower the tyrosine p K_a. Due to sterics within the active site, the proton must either transfer initially to the O(Fe) (a high-energy intermediate), or from another residue over a ∼10 Å distance to reach the O(Cu) atom directly. While the distance between the H⁺ donor (Tyr) and acceptor (O(Cu)) results in a barrier to PT, this separation is critical for the low barrier to O-O cleavage as it enhances backbonding from Fe into the O₂^2- σ* orbital. Thus, PT from Tyr precedes O-O elongation and is rate-limiting, consistent with available kinetic data. The electron transfers from tyrosinate after the barrier via a superexchange pathway provided by the cross-link, generating intermediate P_M. P_M is evaluated using available experimental data. The geometric structure contains an Fe^IV═O that is H-bonded to the Cu^II-OH. The electronic structure is a singlet, where the Fe^IV and Cu^II are antiferromagnetically coupled through the H-bond between the oxo(Fe) and hydroxo(Cu) ligands, while the Cu^II and Tyr^• are ferromagnetically coupled due their delocalization into orthogonal magnetic orbitals on the cross-linked His residue. These findings provide critical insights into the mechanism of efficient O₂ reduction in HCOs, and the nature of the P_M intermediate that couples this reaction to proton pumping.

Figure 1.

Structure of the heme/Cu active site in bovine CcO (PDB 3WG7).

Figure 2.

Current consensus mechanism in HCOs, including the proposed involvement of a peroxo intermediate (I_p) prior to O–O cleavage.

Figure 3.

DFT-optimized structures constructed from the crystal structure of the bovine CcO active site (3WG7) without water molecules included (denoted model {0}), showing the A) peroxo (I_P), and B) ferryl/Y* (P_m) species (i.e. the reactant and product structures in O–O cleavage).

Figure 4.

DFT-optimized structures for the reactant in the H-Bond Assisted mechanism in LS-AN (A) and CcO (B), where the H-bond donor is bound to the Ocu atom of the bridging peroxo ligand. Note that Val243 in {1C}-I_p has been removed for visual clarity.

Figure 5.

Calculated energetics (ΔG, in kcal/mol) in the reaction coordinate for the H-Bond Assisted mechanism in LS-AN (black) and CcO (blue). The experimental barrier in CcO (ΔG^‡ < 12.4 kcal/mol) is given for reference.

Figure 6.

Side (left) and top-down (right) views of the active site, illustrating the two possible ways in which a water molecule may access the bound O₂. The blue circle marks the water location in model {1C}, and the red circle marks the location occupied in model {1F} (note each contains only one H₂O molecule). The gray X in the left figure indicates the region that would provide a direct path from Tyr to the O_cu atom, which is hindered by the His240 and Val243 residues.

Figure 7.

Process of H⁺ transfer from the active site Tyr to the peroxo O_cu atom, mediated by the interstitial water molecule in model {IF}. The upper half of each panel shows a side-on view of the active site, while the lower half shows a top-down view (down the O_Fe-Fe bond). Calculated ΔG values (in kcal/mol, relative to {1F}-I_p) for each optimized species are shown in green, and barriers for each step given in red.

Figure 8.

Calculated energetics (ΔG, in kcal/mol) in the reaction coordinate for the Proton-Initiated mechanism in the synthetic model {LS-AN + PhOH} (black) and the CcO model {1F} (red), which has a water molecule bridging the Tyr and O_Fe atom of the peroxo. The ΔG^‡ < 12.4 kcal/mol experimental barrier in CcO is shown for reference.

Figure 9.

DFT-optimized structure with a H⁺ transferred from an external donor to the O_cu atom, where the Tyr remains protonated.

Figure 10.

MO contours along the singlet surface for the PI mechanism, showing the progressive transfer of an α electron from the Tyr HOMO (marked with a red label) into the O–O σ* (marked with a green label) via the unoccupied α dx²-y² as the O–O bond elongates, after the transition state.

Figure 11.

MO contours along the triplet surface for the PI mechanism, showing the progressive transfer of an α electron from the Tyr HOMO (marked with a red label) into the O–O σ* (marked with a green label) as the O–O bond elongates, after the transition state.

Figure 12.

(left) DFT-optimized structures of {0}-P_M, {1F}-P_M, and {1C}-P_M (as labelled in the figure), representing the possible products generated following O-O cleavage via the Proton-Initiated mechanism. Selected bond distances (marked on the {0}-P_M structure) are given in the text. (right) Localized spins for the Fe^IV, Cu^II, and Tyr^• comprising the singlet and triplet ground states, with the magnetic couplings (J) labelled in the diagram for the singlet (top). The highlighted configurations ({0}-P_M and S=0) are those in closest agreement with available experimental and computational data. Note that Val243 has been removed in the {1F}-P_M structure shown for clarity.

Figure 13.

MOs involved in the superexchange pathway between Fe and Cu, illustrating (A) Fe^IV=O singly occupied orbital character mixed into the Cu^II hole, and (B) Cu^II singly occupied orbital character mixed into a hole on Fe^IV=O. Orbitals labelled in parentheses are doubly occupied.

Figure 14.

Schematic representation of orbital overlap of the cross-linked His donor orbitals with the half-occupied orbitals on Cu^II and Tyr^•, which leads to either ferromagnetic (left) or antiferromagnetic (right) coupling, depending on whether His π character mixes into the Cu LUMO. Note that the orbitals depicted for ferromagnetic coupling represent those shown in Figure 13A and C.

Scheme 1.

Two possible reaction pathways for proton-initiated O–O cleavage, with calculated barriers (in kcal/mol) and H/D KIEs for each step. Energies in each model are relative to the optimized “I_P” structure. The barriers for PT from a nearby amino acid are calculated using transition state theory, assuming a pK_a that is 2 units higher than that of the active site Tyr (see text and SI).