Role of the -PEWY-glutamate in catalysis at the Qo-site of the Cyt bc1 complex

► In mutants at the -PEWY- glutamate, the second electron transfer becomes limiting. ► With kcat ~ 103 s− 1, electron transfer from the distal domain is kinetically incompetent. ► Bifurcated rate at the Qo-site is pH independent below 8, and increases with pK ~ 8.5. ► There is no marked pH-dependence of ES formation over the range < 8 in mutants. ► E295 catalyzes H+ exit and likely acts as a gate for semiquinone migration in the site.