First, oxidized Q binds at site 1 by hydrogen bonding to Tyr144, with charge-neutral His95-Asp196 pair (not shown in Fig. ) also contributing to Q binding and its stabilization (section 5). Next, electron transfer from FeS cluster N2 (orange circle) results in transient formation of anionic SQ (SQ-*). The SQ species is unstable but is indeed formed transiently after first electron transfer from N2. QM-based calculations and simulations show that one-electron reduction of Q does not result in proton transfer from the nearby residues , suggesting that the pKa of SQ is likely to be low and it remains anionic. Non-equilibrium pulling simulations show that the diffusion of anionic SQ in the Q tunnel is energetically expensive . This means that anionic SQ remains trapped at site 1 until reduced by a second electron transfer from N2 (Fig. ). However, the doubly reduced Q (without compensating proton from nearby residues, i.e. Q 2-) is known to be unstable , and hence, two-electron reduction of Q occurs in tandem with its local protonation from a proton donor XH in an energetically favorable manner forming anionic quinol (QH -). The identity of the local proton donor (X) is unclear but Tyr144, which forms a direct hydrogen bond to Q (see section 5), is a possible candidate. Furthermore, the proton donor could also be other active site residues such as His95/Asp196 pair or an ensemble of residues but in all cases a negative charge X -would be generated in the vicinity of the Q binding pocket (see more below).