We next envisage that the negative charge of X-pushes the anionic quinol (QH -) away from site 1 due to strong electrostatic repulsion between the two (ca. 16.6 kcal/mol or ~720 meV with a distance of ca. 5 Å between anionic Tyr and QH -bound at site 1 with ε=4) causing charge separation. This charge separation is important to drive proton pumping, and also in preventing the reverse electron transfer from reduced Q to N2 and coupled (re)protonation reactions. Anionic quinol moves to the Q binding sites closer to the entrance e.g. site 4, which is near the Q tunnel (blue lines) -E channel (red lines) junction (see also Fig. ). The rearrangement of the β1-β2 loop of NDUFS2 is known to occur to accommodate repositioning of Q, as observed in several structures and simulations . Structural changes in the His95 sidechain can also occur , and as proposed earlier may stabilize the anionic state of Tyr144 . The two negative charges in the buried low-dielectric interior of the protein (on X and Q) as well as conformational changes observed in the TMH5-6 loop of ND1 subunit can drive uptake of two protons from the bulk N phase maintaining charge neutrality. The uptake of the first proton (green) occurs to the putative proton loading site (P, marked in purple, Fig. ) stabilizing the negatively charged quinol (QH -) at site 4. This proton is recruited from the N side via the hydrated path(s) identified in structures and simulations (Figs. and).