O2 reactivity: T3 vs Coupled Binuclear Cu (Hc and Tyr) sites
It is important to emphasize that while Hc and Tyr also have two coppers each held in the protein by three His ligands, they have a fundamentally different reactivity with dioxygen. We probed this directly using X-ray absorption spectroscopy (XAS) at the Cu K-edge. In collaborative studies with Prof. Keith Hodgson, we have shown that there is a feature at 8984 eV characteristic of reduced, Cu(I), which is not present in oxidized, Cu(II), sites.20 From figure 7A, deoxy Hc has two reduced Cu’s which are oxidized to two Cu(II)’s in oxy Hc based on the loss of the 8984 eV feature.21 We have characterized a type 2 depleted (T2D) derivative of the MCO’s where the T2 Cu is reversibly removed and remaining is the T3 (and T1) to react with O2.22 From figure 7B, in contrast to Hc and Tyr, the reduced T3 Cu center does not react with O2.20
As a reference we first consider the reversible binding of O2 by Hc. Deoxy Hc has two Cu(I)’s at a distance of ~4.5 Å. This reacts with triplet O2 to generate oxy Hc which has two Cu(II)’s (vide supra) side-on bridged by peroxide at a Cu-Cu distance of 3.6 Å.23 This is an antiferromagnetically (AF) coupled singlet center, thus, this reaction is spin forbidden. A reaction coordinate was generated by systematically varying the distance of the peroxide above the molecular plane and optimizing the rest of the structure.25 From figure 8A, left to right, the structure first butterflies then goes to an asymmetric end-on/side-on bridged structure and then end-on/end-on bridged in the reversible loss of O2. These structures maximize metal-ligand overlap with increasing distances of the oxygen from the copper. From figure 8B, proceeding along this coordinate the peroxide gets less negative and the Cu’s less positive indicating that charge is transferred from peroxide to the two Cu(II)’s. Importantly, the charge on both Cu’s changes at the same rate even in the asymmetric bridged structure (dashed) indicating that O2 binding involves the simultaneous transfer of two electrons.
Figure 8C accounts for the change in spin and total energy along the reaction coordinate. Oxy Hc has a singlet ground state due to AF coupling of the two Cu(II)’s through the π* orbital of the μ-η2:η2 peroxide in the molecular plane. As we proceed along the coordinate the structure becomes butterflied and each Cu(II) interacts with a different π* orbital on the peroxide. This involves orthogonal magnetic orbitals producing a triplet ground state for the butterflied structure. The peroxide can then directly transfer one electron of the same spin to each Cu leading to triplet dioxygen which is further energetically stabilized by single center exchange.24
Importantly, O2 binding to Hc is found to be exothermic by 3 kcal/mol.25 This is contrasted to O2 binding to the deoxy T3 center of T2D laccase in figure 9, where O2 binding is found to be uphill by 6 kcal/mol. As shown in figure 10 the origin of this 10 kcal/mol destabilization of O2 binding relative to Hc reflects the relative stabilization of the deoxy T3 structure in the MCO protein environment. The deoxy potential energy surfaces in figure 10 were obtained by geometry optimizing the reduced Hc and T3 sites with their respective protein constraints imposed on the deoxy structures. From figure 10, the deoxy T3 center (red) is 7 kcal/mol lower in energy than deoxy Hc (blue) and has an equilibrium Cu(I)-Cu(I) distance of 6.5 Å, in contrast to the 4.2 Å optimized distance of deoxy Hc. This decrease in energy of the deoxy T3 center dominantly reflects the decrease in electrostatic repulsion of the two Cu(I)’s in the low dielectric of the protein (dashed blue) and an additional smaller contribution due to a decrease in sterric interactions of the three His ligands between Cu centers which are eclipsed in the T3 center and staggered in Hc (difference between dashed red and dashed blue). Thus the lack of O2 reactivity of the deoxy T3 site reflects its electrostatic and structural stabilization at its long 6.5 Å Cu-Cu distance. From figure 11 the large structural differences between the coupled binuclear site in Hc and the T3 site in the MCO’s relate to very different structural constraints in the two protein environments. The binuclear cuprous site of deoxy Hc is kept at its 4.2 Å Cu-Cu distance due to the constraint associated with two His ligands, one on each Cu, each deriving from a different helix bundle held together by a salt bridge. Alternatively in the T3 Cu center the two Cu(I)’s are kept at an electrostatically stable distance of 6.5 Å by two sets of two His ligands, where each set is from an H-X-H bridging motif that is on a loop extending from a β sheet, leaving the Cu(I)’s relatively unconstrained.
In summary deoxy Hc is electrostatically destabilized to react with O2 to form oxy Hc and this can be cooperatively regulated by changing the Cu(I)-Cu(I) distance in tensed and relaxed protein quaternary structures,26 while the reduced T3 site in the MCOs is electrostatically stable and does not react with O2 when the T2 Cu is not present.