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

Abstract We re-examine the pH dependence of partial processes of ubihydroquinone (QH 2 ) turnover in Glu-295 mutants in Rhodobacter sphaeroides to clarify the mechanistic role. In more crippled mutants, the bell-shaped pH profile of wildtype was replaced by dependence on a single p K at ~ 8.5 favoring electron transfer. Loss of the p K at 6.5 reflects a change in the rate-limiting step from the first to the second electron transfer. Over the range of pH 6–8, no major pH dependence of formation of the initial reaction complex was seen, and the rates of bypass reactions were similar to the wildtype. Occupancy of the Q o -site by semiquinone (SQ) was similar in the wildtype and the Glu → Trp mutant. Since heme b L is initially oxidized in the latter, the bifurcated reaction can still occur, allowing estimation of an empirical rate constant 3  s − 1 for reduction of heme b L by SQ from the domain distal from heme b L , a value 1000-fold smaller than that expected from distance. If the p K ~ 8.5 in mutant strains is due to deprotonation of the neutral semiquinone, with Q •− as electron donor to heme b L , then in wildtype this low value would preclude mechanisms for normal flux in which semiquinone is constrained to this domain. A kinetic model in which Glu-295 catalyzes H + transfer from QH•, and delivery of the H + to exit channel(s) by rotational displacement, and facilitates rapid electron transfer from SQ to heme b L by allowing Q •− to move closer to the heme, accounts well for the observations.