Network analysis of a proposed exit pathway for protons to the P-side of cytochrome c oxidase

Cytochrome c Oxidase (CcO) reduces O₂, the terminal electron acceptor, to water in the aerobic, respiratory electron transport chain. The energy released by O₂ reductions is stored by removing eight protons from the high pH, N-side, of the membrane with four used for chemistry in the active site and four pumped to the low pH, P-side. The proton transfers must occur along controllable proton pathways that prevent energy dissipating movement towards the N-side. The CcO N-side has well established D- and K-channels to deliver protons to the protein interior. The P-side has a buried core of hydrogen-bonded protonatable residues designated the Proton Loading Site cluster (PLS cluster) and many protonatable residues on the P-side surface, providing no obvious unique exit. Hydrogen bond pathways were identified in Molecular Dynamics (MD) trajectories of Rb. sphaeroides CcO prepared in the P_R state with the heme a₃ propionate and Glu286 in different protonation states. Grand Canonical Monte Carlo sampling of water locations, polar proton positions and residue protonation states in trajectory snapshots identify a limited number of water mediated, proton paths from PLS cluster to the surface via a (P-exit) cluster of residues. Key P-exit residues include His93, Ser168, Thr100 and Asn96. The hydrogen bonds between PLS cluster and P-exit clusters are mediated by a water wire in a cavity centered near Thr100, whose hydration can be interrupted by a hydrophobic pair, Leu255B (near Cu_A) and Ile99. Connections between the D channel and PLS via Glu286 are controlled by a second, variably hydrated cavity. SIGNIFICANCE STATEMENT: Cytochrome C oxidase plays a crucial role in cellular respiration and energy generation. It reduces O₂ to water and uses the released free energy to move protons across mitochondrial and bacterial cell membranes adding to the essential electrochemical gradient. Energy storage requires that protons are taken up from the high pH, N-side and released to the low pH, P-side of the membrane. We identify a potential proton exit from a buried cluster of polar residues (the proton loading site) to the P-side of CcO via paths made up of waters and conserved residues. Two water cavities connect the proton exit pathway to the surface only when hydrated. Changing the degree of hydration may control otherwise energetically favorable proton backflow from the P-side.