Dynamic water networks in cytochrome C oxidase from Paracoccus denitrificans investigated by molecular dynamics simulations

Abstract

We present a molecular dynamics study of cytochrome c oxidase from Paracoccus denitrificans in the fully oxidized state, embedded in a fully hydrated dimyristoylphosphatidylcholine lipid bilayer membrane. Parallel simulations with different levels of protein hydration, 1.125 ns each in length, were carried out under conditions of constant temperature and pressure using three-dimensional periodic boundary conditions and full electrostatics to investigate the distribution and dynamics of water molecules and their corresponding hydrogen-bonded networks inside cytochrome c oxidase. The majority of the water molecules had residence times shorter than 100 ps, but a few water molecules are fixed inside the protein for up to 1.125 ns. The hydrogen-bonded network in cytochrome c oxidase is not uniformly distributed, and the degree of water arrangement is variable. The average number of solvent sites in the proton-conducting K- and D-pathways was determined. In contrast to single water files in narrow geometries we observe significant diffusion of individual water molecules along these pathways. The highly fluctuating hydrogen-bonded networks, combined with the significant diffusion of individual water molecules, provide a basis for the transfer of protons in cytochrome c oxidase, therefore leading to a better understanding of the mechanism of proton pumping.

FIGURE 1

(A) Partial atomic charges for the fully oxidized heme a/heme a₃ derived from quantum chemical calculations. (B) Partial atomic charges for Tyr-280–His-276 crosslink (charges for His-276 are listed in C); (C) partial atomic charges for the histidine ligands to Cu_B. The charges on His-326 were restrained to be identical to those of His-325 and therefore not shown. (D) Partial atomic charges for the histidine ligand to Fe atom of heme a₃. (E) Partial atomic charges for histidine ligands to Fe atom of heme a; the two His ligands carry the same charges.

FIGURE 2

Simulation system setup (side view): two-subunits of cytochrome c oxidase embedded in a DMPC membrane solvated by a 100 mM NaCl aqueous salt solution (the Na⁺ ions are yellow and the Cl⁻ ions are blue). The snapshot was taken at the beginning of the molecular dynamics production phase after 575 ps of equilibration for the W12 set.

FIGURE 3

Root mean-square deviation (RMSD) relative to the x-ray structure as a function of time, calculated over all backbone atoms (black line), side-chain atoms (gray line), and heme a/a₃ atoms (light gray). (A) RMSD from the W12 set of coordinates; (B) RMSD from the W8 set.

FIGURE 4

RMSF of the backbone atoms calculated from the molecular dynamics trajectories at 1–45 ps (black line), 450–495 ps (red line), 1080–1125 ps (green line), and from the experimental B-factor (blue line). All values are averaged over the individual amino acids. (a) RMSF from the W12 set. (b) RMSF from the W8 set.

FIGURE 5

RMSF of the backbone atoms calculated from the molecular dynamics trajectories for the W12 and W8 sets of water molecules during 1–45 ps (a), 450–495 ps (b), and 1080–1125 ps (c). All values are averaged over the individual amino acids; the backbone atoms from the W12 set of coordinates are shown in black; the backbone atoms from the W8 set of coordinates are shown in red.

FIGURE 6

The distribution of water molecules in the COX during the simulation.

FIGURE 7

Multiple configurations of selected residues and water molecules in the K-pathway for the W12 (A) and W8 (B) set of coordinates observed from MD trajectories. Initial coordinates are shown with the thick licorice. Positions of the selected residues after 1125 ps are shown with the thin licorice. Snapshots for selected water molecules are taken after 225, 450, 675, 900, and 1125 ps of simulations. For the W12 set of coordinates water molecule W_S6 (structural water) is represented in blue, W_S9 in yellow, W_S69 in red, W_S81 in rose, W_G7 (internal GRID water) in light green, W_G149 in pink, and W_G161 in magenta. For the W8 set of coordinates water molecule W_S6 is represented in blue, W_S69 in red, W_S88 in yellow, W_G13 in rose, W_G188 in light green, W_G194 in gold, W_G379 in pink, W_G491 in magenta, and W₂6484 in orange.

FIGURE 8

Multiple configurations of selected residues and water molecules in the D-pathway for the W12 (A) and W8 (B) set of coordinates observed from MD trajectories. Initial coordinates are shown with the thick licorice. Positions of the selected residues after 1125 ps of MD run are shown with the thin licorice. Snapshots for selected water molecules are taken after 225, 450, 675, 900, and 1125 ps of simulations. For the W12 set of coordinates water molecule W_S38 is represented in blue, W_S40 in yellow, W_S80 in red, W_G8 in light green, W_G12 in gold, W_G80 in light blue, W_G131 in magenta, and W_G144 in pink. For the W8 set of coordinates water molecule W_S3 is represented in rose, W_S38 in blue, W_G9 in light blue, W_G11 in orange, W_G180 in light green, W_G198 in magenta, W_G220 in green, W_G238 in red, W_G540 in gray, and W₁232 in black.

FIGURE 9

Multiple configurations of selected residues and water molecules close to the heme a/heme a₃–Cu_B region for the W12 (A) and W8 (B) set of coordinates observed from MD trajectories. Initial coordinates are shown with the thick licorice. Positions of the selected residues after 1125 ps of MD run are shown with the thin licorice. Snapshots for selected water molecules are taken after 225, 450, 675, 900, and 1125 ps of simulations. For the W12 set of coordinates water molecule W_S13 is represented in blue, W_S69 in red, W_G5 in yellow, W_G7 in green, W_G30 in pink, W_G144 in magenta, W_G173 in orange, W₄172 in gold, and W₄214 in rose. For the W8 set of coordinates water molecule W_S13 is represented in blue, W_S14 in yellow, W_S69 in red, W_G31 in light green, W_G42 in orange, W_G46 in magenta, W_G51 in pink, W_G157 in green, W_G188 in rose, W_G189 in black, and W_G453 in gold.