Dewetting transitions coupled to K-channel activation in cytochrome c oxidase

Abstract

Cytochrome c oxidase (CcO) drives aerobic respiratory chains in all organisms by transducing the free energy from oxygen reduction into an electrochemical proton gradient across a biological membrane. CcO employs the so-called D- and K-channels for proton uptake, but the molecular mechanism for activation of the K-channel has remained elusive for decades. We show here by combining large-scale atomistic molecular simulations with graph-theoretical water network analysis, and hybrid quantum/classical (QM/MM) free energy calculations, that the K-channel is activated by formation of a reactive oxidized intermediate in the binuclear heme a ₃/Cu_B active site. This state induces electrostatic, hydration, and conformational changes that lower the barrier for proton transfer along the K-channel by dewetting pathways that connect the D-channel with the active site. Our combined results reconcile previous experimental findings and indicate that water dynamics plays a decisive role in the proton pumping machinery in CcO.

Fig. 1. Structure and function of cytochrome c oxidase (CcO). O₂ reduction to H₂O drives the electron transfers (blue arrow) to the binuclear centre (BNC: heme a₃/Cu_B) and proton transfers (red arrow) to the BNC for oxygen chemistry and for pumping of protons to the positively charged (P) side across the membrane. Protons are taken up from the negatively charged (N) side of the membrane through conserved D- and K-channels, terminating at Glu-242 and Tyr-244, respectively. Water molecules occupying the channels are shown as red spheres. Hemes are shown in purple and the copper centres are depicted in orange. Inset: close-up of the structure and proton transfer (pT)/electron transfer (eT) pathways between Glu-242, Lys-319, the proton loading site (PLS),– heme a, and heme a₃/Cu_B. (B) Catalytic cycle of CcO showing the BNC and the catalytic intermediates. Protons (H⁺) marked in blue and red refer to chemical protons taken up from the D- and K-channels, respectively. Protons pumped across the membrane are indicated in black.

Fig. 2. Lys-319 sidechain dynamics, water occupancy in the K-channel above Lys-319 and K-channel volume (in ų) from 500 ns of MD simulations. (A) Distances between Lys-319 and Tyr-244 from simulations of P_M, P_R, O_H, and O_H,R states. (B) Water occupancies in the K-channel between Lys-319 and Tyr-244 from simulations of P_M, P_R, O_H, and O_H,R states. Modal water occupancies are indicated as dotted lines. (C) Water accessible volume in the K-channel for the P_R and O_H,R states.

Fig. 3. Longest non-bonded connectivity along the shortest path connecting the proton donor and proton acceptor, ζ, calculated using Dijkstra's algorithm in graph theory. (A) Water-mediated connectivity from Glu-242 to Cu_B in P_M, P_R, O_H, and O_H,R states. Thresholds for hydrogen-bonding connectivity are shown in magenta (2.5 Å) and yellow (4.0 Å) dashed lines. (B) Water-mediated connectivity from Lys-319 to Tyr-244 in P_M, P_R, O_H, and O_H,R states. (C) Water occupancy in the non-polar cavity above Glu-242 in P_M, P_R, O_H, and O_H,R states.

Fig. 4. QM/MM-MD free energy profiles for proton transfer from Glu-242 to Cu_B in the P_R state and from Lys-319 to Tyr-244 in the P_R and O_H,R states. Lighter traces indicate the statistical error for the free energy profile. Snapshots of the reactants and products for the proton transfer profiles from Glu-242 to Cu_B in the P_R state and from Lys-319 to Tyr-244 in the O_H,R state shown in the bottom panel (see ESI-Fig. 12 for more snapshots†).