Density Functional Study for the Bridged Dinuclear Center Based on a High-Resolution X‑ray Crystal Structure of ba₃ Cytochrome c Oxidase from Thermus thermophilus

Strong electron density for a peroxide type dioxygen species bridging the Fe_a3 and Cu_B dinuclear center (DNC) was observed in the high-resolution (1.8 Å) X-ray crystal structures (PDB entries 3S8G and 3S8F) of ba₃ cytochrome c oxidase (CcO) from Thermus thermophilus. The crystals represent the as-isolated X-ray photoreduced CcO structures. The bridging peroxide was proposed to arise from the recombination of two radiation-produced HO^• radicals formed either very near to or even in the space between the two metals of the DNC. It is unclear whether this peroxide species is in the O₂^2–, O₂^•–, HO₂^–, or the H₂O₂ form and what is the detailed electronic structure and binding geometry including the DNC. In order to answer what form of this dioxygen species was observed in the DNC of the 1.8 Å X-ray CcO crystal structure (3S8G), we have applied broken-symmetry density functional theory (BS-DFT) geometric and energetic calculations (using OLYP potential) on large DNC cluster models with different Fe_a3–Cu_B oxidation and spin states and with O₂^2–, O₂^•–, HO₂^–, or H₂O₂ in the bridging position. By comparing the DFT optimized geometries with the X-ray crystal structure (3S8G), we propose that the bridging peroxide is HO₂^–. The X-ray crystal structure is likely to represent the superposition of the Fe_a3²⁺–(HO₂^–)–Cu_B⁺ DNC’s in different states (Fe²⁺ in low spin (LS), intermediate spin (IS), or high spin (HS)) with the majority species having the proton of the HO₂^– residing on the oxygen atom (O1) which is closer to the Fe_a3²⁺ site in the Fe_a3²⁺–(HO–O)⁻–Cu_B⁺ conformation. Our calculations show that the side chain of Tyr237 is likely trapped in the deprotonated Tyr237^– anion form in the 3S8G X-ray crystal structure.