- Density functional theory (1) (remove)
- Electrostatic and quantum chemical investigation of the proton pumping mechanism of cytochrome c oxidase (2008)
- Cytochrome c oxidase is a crucial enzyme in the respiratory chain. It catalyzes the reduction of oxygen to water and utilizes the free energy of the reduction reaction for proton pumping across the inner-mitochondrial membrane, a process which results in a membrane electrochemical proton gradient. For each oxygen molecule, eight protons are taken up from the matrix of the mitochondria. Four protons together with four electrons are required to reduce oxygen to water at the Fea3 -CuB binuclear center and another four protons are translocated across the membrane. Although several high resolution structures have been solved for this enzyme, the molecular mechanism of the proton pumping and electron transfer is not understood. Recent studies on the cytochrome c oxidase CuB center suggested deprotonation of the CuB bound imidazole ring of histidine (His291 in mammalian cytochrome c oxidase or His334 in Rhodobacter sphaeroides cytochrome c oxidase) as a key element in the proton pumping mechanism. The central feature of this proposed mechanism is that the pKa value of the imidazole significantly lowered depending on the redox state of the metals in the binuclear center. The energetic feasibility of this mechanism is tested in this work. To comprehend the role of the CuB bound histidines in the reaction mechanism of cytochrome c oxidase, density functional theory is used in combination with continuum electrostatics to calculate the pKa values of these imidazole rings in the aqueous solution as well as in the protein. The pKa values of His334, His333 and H2 O molecule are calculated both in oxidized and reduced state of CuB center. The Finite Difference Poisson Boltzmann (FDPB) method and the conductor-like polarizable continuum model (C-PCM) are used to determine the solvation free energies in aqueous solution. All possible protonation equilibrium reactions in the CuB center are studied to understand the deprotonation reactions of the bound H2 O molecule, His333 and His334. In aqueous solution, pKa values of 15.2, 15.9 and 7.4 were obtained for deprotonation of His334, His333 and H2O respectively. These pKa values in aqueous solution show that His334 and His333 are likely to be protonated at physiological pH. The protein environment shifts the pKa values of the CuB ligands to even higher values in the range between 15 to 60. These pKa values of CuB ligands are significantly higher compared to aqueous solution. The high pKa values show that His334 is protonated during all steps of the catalytic cycle and demonstrate that the Fe and Cu ion oxidation states do not lower the pKa values of CuB ligands and involved in shifting the pKa values of CuB ligands to higher values. These results are incompatible with the proposed role of His334 as a key element in the pumping mechanism. According to the pKa values, the proton pumping model as suggested by Stuchebrukhov might not be possible with the involvement of His334. The pKa values of the His333 in the CuB center are always shifted to higher values both in the reduced and in the oxidized state of the CuB center. The pKa values of His333 show that this residue is likely to be protonated in the protein and an involvement in the reaction mechanism of cytochrome c oxidase can therefore be ruled out.