The role of active site residues in protonation of Q deserves further discussion. We note that computer simulation data supports the role of catalytic triad, Tyr144, His95 and Asp196 in proton transfer to Q upon its reduction. However, what remains unclear is that if two-electron reduction of Q results in its single or double protonation, leading to the formation of QH -or QH2, respectively. Quantitative free energy simulations combined with spectroscopic experiments would be required to resolve this issue. However, current data suggests that a single proton transfer from proton donor X forming QH -is sufficient to create charge imbalance in the Q cavity, which can drive proton pumping (see above). This mechanistic picture is supported by Tyr144Phe mutation data, which suggests that complex I tolerates the absence of Tyr144 (section 5). In that case, His95/Asp196 pair may take the role of proton donor X, and provide the required proton for Q reduction leading to the formation of anionic quinol (QH - ) and not QH2. Interestingly, Asp196Asn and His95Arg (59) mutations, both of which are expected to strip off the role His/Asp pair as a proton donor X, shows that the enzyme is still partially active. In these cases, Tyr144 supposedly provides the required proton upon two electron reduction of Q. All in all, these data provide support to the possibility that a singly protonated quinol (QH -) is produced after two electron transfer from N2, together with anionic X. Fig. . Proton pumping model of complex I. See text for description.