Next, an exergonic substrate proton (yellow) transfer occurs to anionic quinol (also utilizing the pathway(s) described in Figs. and), breaking the charge balance between the negatively charged Q and the first pumped proton on the proton loading site. The pumped proton is driven towards the membrane domain via the E channel. Protonation of anionic quinol results in neutral QH2, which leaves the Q tunnel. The PLS is reloaded with a second proton (green) driven and stabilized by the negative charge of X-, which may have diffused closer to the E-channel/Q tunnel junction due to charge-charge rearrangements in titratable residues lining the Q tunnel. Indeed, structural data on complex I from Y. lipolytica in turnover conditions reveal conformational changes that lead to charge-charge rearrangements in the vicinity of the Q tunnel . Departure of QH2 results in Q tunnel hydration (as observed in computer simulations ) and the second substrate proton is transferred eventually to nullify the negative charge of X. This second exergonic step (neutralization of anionic X) by a second substrate proton (yellow) from the N side, which results in the release of the second pumped proton from the PLS towards the E channel. Subsequently, the tunnel gets dehydrated again and a new Q molecule binds near N2 site (site 1) to restart the cycle. We note that the reduction (and local protonation) of Q to QH -at site 1, but its protonation from the N phase of the membrane at the site near E channel/Q tunnel junction (site 4) is in agreement with the two-state model proposed by , and also with the suggestion that site 1 is a low potential (Em) region, whereas site 4 is a high potential region .