DFT/Electrostatic Calculations of pK_a Values in Cytochrome c Oxidase

Abstract

Using classical electrostatic calculations, earlier we examined the dependence of the protonation state of bovine cytochrome c oxidase (CcO) on its redox state. Based on these calculations, we have proposed a model of CcO proton pumping that involves His291, one of the Cu(B) histidine ligands, which was found to respond to redox changes of the enzyme Fe(a)(3)-Cu(B) catalytic center. In this work, we employ combined density functional and continuum electrostatic calculations to evaluate the pK(a)() values of His291 and Glu242, two key residues of the model. The pK(a) values are calculated for different redox states of the enzyme, and the influence of different factors on the pK(a)'s is analyzed in detail. The calculated pK(a)() values of Glu242 are between 9.4 and 12.0, depending on the redox state of the protein, which is in excellent agreement with recent experimental measurements. Assuming the reduced state of heme a(3), His291 of the oxidized Cu(B) center possesses a pK(a)() between 2.1 and 4.0, while His291 of the reduced Cu(B) center has a pK(a) above 17. The obtained results support the proposal that the His291 ligand of the Cu(B) center in CcO is a proton pump element.

Dielectric relaxation of cytochrome c oxidase: Comparison of the microscopic and continuum models

“…More generally, one finds that for the commonly used continuum parameters, 3,21,40,44 the sum of the probe and atomic radius significantly exceeds the equilibrium interatomic distance between given protein atom and water oxygen observed in the x-ray data. The reason for this is that internal water molecules form strong hydrogen bonds with protein structure, 6,7 but commonly used parameters do not properly take into account this effect.…”

“…Thus, the free energy change due to introduced charges consists of two parts: The protein field term, representing the work needed to transfer charges ⌬q i from infinity to the corresponding atomic positions of the solute at fixed ͑equilibrium͒ nuclear positions of the latter, and the reaction-field term, representing the free energy change due to the relaxation of the equilibrium atomic positions in response to the introduced charges. In the literature, ⌬G prot and ⌬G rf are also called as the protein field and Born term, 3,21,35,36 static and relaxation term, 14,15,17 or as the interaction and self-energy terms, 4,25,27 respectively. In this paper, we will be interested only in ⌬G rf term which reflects the dielectric relaxation of the protein medium.…”

“…The most computationally efficient treatments of electrostatic interactions and estimation of pK a values in proteins are based on the continuum models, [1][2][3] in which the effective dielectric constants for the protein are used. The results obtained with such models are very sensitive to the value of the dielectric constant; however, the optimal value of this parameter for a particular protein is difficult to choose or justify.…”

“…The values of such parameters are often different in different studies, which is one of the reasons for the differences in the pK a calculations of the enzyme. 3,21,22 The key parameter appears to be the dielectric constant of the enzyme water cavities cav , about which very little is known. Although it is well recognized that different cavities may have different dielectric properties ͑e.g., size dependent͒, it is important to know typical magnitudes of their dielectric relaxation parameters.…”

“…This residue is a key element of a recently proposed proton pumping mechanism of the enzyme. 3,21 The dielectric constant of the protein p and that of an important water cavity cav in the vicinity of the catalytic center ͑see Fig. 1͒ are determined as adjustable parameters which reproduce the microscopic reaction-field energy of charging ͑or deprotonation͒ obtained in MD simulations of the dry protein and the protein with internal water.…”