We now shift the focus from Q reductase domain to membrane domain of complex I, which catalyzes proton transfer to the P side of the membrane. Historically, each of the three antiporter-like subunits of complex I (ND2, ND4 and ND5 in membrane domain, Fig. ) have been assumed to take up protons from the N side and to release them to the P side in coupling with the redox reaction in the Q tunnel. Structural, biochemical and simulation data have suggested possible proton transfer pathways that comprise conserved polar/charged amino acid residues and water molecules. The pathways from the N side of the membrane to the interior of the antiporter-like subunits and pathways contributing to the central hydrophilic axis have become clear in the recent high-resolution structures and multiscale MD simulations . Point mutations introduced in antiporter-like subunits (section 1) have supported the role of each subunit in energy conservation by pumping protons to the P side of the membrane. However, pathways from the central axis leading to the P side have been difficult to discern in two of the three antiporter-like subunits. Neither structures nor simulations have revealed a consistent P side pathway in ND2 and ND4 subunits, where water occupancy has been scarce (Fig. ). Only in the terminal ND5 subunit a hydrated path is consistently observed based on structural and simulation data, because of its unique architecture, additional conserved charged residues, and proximity to the membrane (16, 75) (Fig. ). In contrast, for the ND2 and ND4 subunits, an arrangement of hydrophobic residues appears to block connections to the P side. The location of the P side water path in ND5 is between TMH15 and the conserved broken TMH12. The equivalent water path in ND2 and ND4 should therefore be between TMH12 and TMH6 of the next subunit along. However, these environments are more hydrophobic than the ND5 environment, which makes access to solvent much more difficult. All in all, the three subunits are clearly not equivalent , even though they are often grouped together collectively as the antiporter-like subunits. ). The proposed proton-loading site (PLS, purple) is situated at the junction of the E channel (red) and Q tunnel (blue) next to the Q binding site 4 (see also Fig. ). Uptake of substrate protons (yellow) that neutralize the anionic intermediates. The antiporter-like subunits also take up protons from the N side (green) and localize them on to the central axis. PLS protons injected into the E channel (green) electrostatically pushes the pumped protons loaded on the central axis of the membrane-bound subunits to be released to the P side via ND5 exit. Note in panel B, several putative proton transfer pathways for pumped and substrate protons are marked and do not signify pumping stoichiometry. ND5 consists of a unique functional element, histidine switch, where a conserved histidine undergoes conformational transition between two positions (marked as HA and HB). The red dotted lines mark uncertain P side paths in ND2 and ND4 subunits.