A broken-symmetry density functional study of structures, energies, and protonation states along the catalytic O–O bond cleavage pathway in ba3 cytochrome c oxidase from Thermus thermophilus

Abstract

Broken-symmetry density functional calculations have been performed on the [Fe_a3, Cu_B] dinuclear center (DNC) of ba₃ cytochrome c oxidase from Thermus thermophilus in the states of [Fe_a3³⁺–(HO₂)⁻–Cu_B²⁺, Tyr237⁻] and [Fe_a3⁴⁺O²⁻, OH⁻–Cu_B²⁺, Tyr237˙], using both PW91-D3 and OLYP-D3 functionals. Tyr237 is a special tyrosine cross-linked to His233, a ligand of Cu_B. The calculations have shown that the DNC in these states strongly favors the protonation of His376, which is above propionate-A, but not of the carboxylate group of propionate-A. The energies of the structures obtained by constrained geometry optimizations along the O–O bond cleavage pathway between [Fe_a3³⁺–(O–OH)⁻–Cu_B²⁺, Tyr237⁻] and [Fe_a3⁴⁺O²⁻⋯HO⁻–Cu_B²⁺, Tyr237˙] have also been calculated. The transition of [Fe_a3³⁺–(O–OH)⁻–Cu_B²⁺, Tyr237⁻] → [Fe_a3⁴⁺O²⁻⋯HO⁻–Cu_B²⁺, Tyr237˙] shows a very small barrier, which is less than 3.0/2.0 kcal mol⁻¹ in PW91-D3/OLYP-D3 calculations. The protonation state of His376 does not affect this O–O cleavage barrier. The rate limiting step of the transition from state A (in which O₂ binds to Fe_a3²⁺) to state P_M ([Fe_a3⁴⁺O²⁻, OH⁻–Cu_B²⁺, Tyr237˙], where the O–O bond is cleaved) in the catalytic cycle is, therefore, the proton transfer originating from Tyr237 to O–O to form the hydroperoxo [Fe_a3³⁺–(O–OH)⁻–Cu_B²⁺, Tyr237⁻] state. The importance of His376 in proton uptake and the function of propionate-A/neutral-Asp372 as a gate to prevent the proton from back-flowing to the DNC are also shown.