Effect of a pH Gradient on the Protonation States of Cytochrome c Oxidase: A Continuum Electrostatics Study

Abstract

Cytochrome c oxidase (CcO) couples the reduction of dioxygen to water with transmembrane proton pumping, which leads to the generation of an electrochemical gradient. In this study we analyze how one of the components of the electrochemical gradient, the difference in pH across the membrane, or ΔpH, influences the protonation states of residues in CcO. We modified our continuum electrostatics/Monte Carlo (CE/MC) method in order to include the ΔpH and applied it to the study of CcO, in what is, to our best knowledge, the first CE/MC study of CcO in the presence of a pH gradient. The inclusion of a transmembrane pH gradient allows for the identification of residues whose titration behavior depends on the pH on both sides of the membrane. Among the several residues with unusual titration profiles, three are well-known key residues in the proton transfer process of CcO: E286I, Y288I, and K362I. All three residues have been previously identified as being critical for the catalytic or proton pumping functions of CcO. Our results suggest that when the pH gradient increases, these residues may be part of a regulatory mechanism to stem the proton flow.

Increasing the Realism of in Silico pHLIP Peptide Models with a Novel pH Gradient CpHMD Method

“…pHRE is an enhanced sampling method extended from the CpHMD-L methodology, ,,− which can be described in three modules: a Poisson–Boltzmann/Monte Carlo (PB/MC) step from which new protonation states are generated for titrable groups using PB-derived free energy terms; a solvent relaxation step where solvent molecules (SPC water model) are allowed to adapt to the new protonation states; and a final molecular mechanics/molecular dynamics (MM/MD) production step where new conformations are sampled. The Baptista’s PB/MC implementation of the pH gradient was integrated into our pHRE methodology. In this setup, the membrane center defines the border between the two regions with different pH values.…”

“…In the CpHMD simulations setup, the pH gradient is defined by two user-specified parameters, the pH and the ΔpH. These are used to calculate pH in and pH out from pH ± (ΔpH/2) . Regarding the non pH gradient simulations, all titrating residues are exposed to a single proton bath at a given pH value in the previously established pH range.…”

“…Monte Carlo (MC) calculations sample the protonation states of all titrating residues using a modified version of the PETIT program that implements the pH gradient setup, as explained in the cited protocol . In this new version, the program requires a preassignment of each titrating site to one of two proton baths, each with a distinct pH value, representing the inner monolayer (pH in ) or the outer (pH out ) monolayer. , The site assignment was static and required a previous insertion analysis, for each titrating residue using the unrestrained CpHMD equilibration data, to determine which monolayer was being populated on average to choose the correct proton bath. The analysis was done for both peptides, where Asp-25 ( wt ) and Asp-19 (Var3) assignment needed to be more cautious since their close position to the membrane center (average position of all P atoms in the membrane normal) allows crossing between both monolayers.…”

Development of constant-pH simulation methods in implicit solvent and applications in biomolecular systems

“…Conversely, improving the molecular surface boundaries and using the finite element method, Sakalli & Knapp impressively obtained the smallest RMSD for lysozyme: 0.18. Multi approaches with a good description of proton isomerism have also been reported in the literature (Magalhães et al 2017). In addition, a practical detailed information for the PB simulation protocols is given in this paper, which may be useful to the reader.…”

Determination of proton concentration at cardiolipin-containing membrane interfaces and its relation with the peroxidase activity of cytochromec

“…The peroxidase activity of cyt c increases upon interaction with CL, resulting in CL oxidation and subsequent apoptosis execution via cyt c permeabilization to the cytosol 19. Recently, it has been suggested that protein activities are affected by the pH gradient across biological membranes 20. The peroxidase activity of yeast cyt c adsorbed onto kaolinite was enhanced remarkably by decreasing the pH value below 4 21.…”