A Protonated Water Cluster as a Transient Proton‐Loading Site in Cytochrome c Oxidase

Abstract

Cytochrome c oxidase (CcO) is a redox-driven proton pump that powers aerobic respiratory chains. We show here by multi-scale molecular simulations that a protonated water cluster near the active site is likely to serve as the transient proton-loading site (PLS) that stores a proton during the pumping process. The pKa of this water cluster is sensitive to the redox states of the enzyme, showing distinct similarities to other energy converting proton pumps.

Mitochondrial cytochrome c oxidase: catalysis, coupling and controversies

“…This trap site has not been identified experimentally, but the general consensus is that it involves the protonatable amino acids and water molecules close to the bound Mg 2+ ion' above the BNC. Quite possibly, the proton is shared between several groups in this region (81), lowering its energy and perhaps explaining its lack of experimental detection. The positively-charged trap site electrostatically lowers the energy required to transfer the negative electron into the BNC, but is positioned such that the proton cannot act as a substrate proton for subsequent water formation.…”

“…However, the detailed order of the other electron and proton transfer steps still requires further definition and understanding. Also outstanding is the possible additional function of the K channel as a dielectric channel in some of the reaction steps (82) as well as the nature of the proton trap site which has to date defied experimental identification, though again simulations have provided support for its location around the Mg ++ site (81).…”

A common coupling mechanism for A-type heme-copper oxidases from bacteria to mitochondria

“…Although the D-channel route into the proton trap is most likely as described above, the path from the trap into the P phase remains generally unresolved. The likely proton trap region is "above" the BNC and close to the bound Mg 2+ /Mn 2+ site (16,62). Protonic connectivity between the trap and P phase may be made through the relatively hydrated "top" region of the H-channel, possibly linked via the conserved pair of arginine ligands to the heme a propionates.…”

Electronation‐dependent structural change at the proton exit side of cytochrome c oxidase as revealed by site‐directed fluorescence labeling

“…An open question is the activation mechanism of the K‐channel, since, in contrast to the D‐channel, a continuous water wire in the K‐channel from the surface to the BNC is absent . Conformational changes of key residues and channel wetting changes in the different states of the catalytic cycle have been implicated in several theoretical studies to facilitate proton transport ; however, experimental evidence is lacking. To this end, we show that at the N‐side in the vicinity of the K‐channel entrance in position 301, which is located in the loop between helix 6 and helix 7 (Fig.…”

“…For this purpose, the so‐called proton‐loading site serves as a transient storage site for the pumped proton until electron transfer to the catalytic site causes C c O to eject them . The location of this site presumably is a highly conserved water cluster with variable p K A close to the heme a 3 propionic acid groups toward the P‐side of the membrane . EPR experimental results have highlighted the role of the Mg 2+ site in the translocation of water and proton pump mechanism toward the P‐side of the membrane .…”