Ubiquinone pair in the Q(o) site central to the primary energy conversion reactions of cytochrome bc(1) complex

The mechanistic heart of the ubihydroquinone-cytochrome c oxidoreductase (cyt bet complex) is the catalytic oxidation of ubihydroquinone (QH(2)) at the Q(o) site. QH(2) oxidation is initiated by ferri-cyt c, mediated by the cyt c(1) and [2Fe-2S] cluster of the cytochrome bc(1) complex. QH(2) oxidation in turn drives transmembrane electronic charge separation through two b-type hemes to another ubiquinone (Q) at the Q(i) site. In earlier studies, residues F144 and G158 of the b-heme containing polypeptide of the Rhodobacter capsulatus cyt bc(1) complex were shown to be influential in Q(o) site function. In the present study, F144 and G158 have each been singly substituted by neutral residues and the dissociation constants measured for both Q and QH(2) at each of the strong and weak binding Q(o) site domains (Q(os) and Q(ow)). Various substitutions at F144 or G158 were found to weaken the affinities for Q and QH(2) at both the Q(os) and Q(ow) domains variably from zero to beyond 10(3)-fold. This produced a family of Q(o) sites with Q(os) and Q(ow) domain occupancies ranging from nearly full to nearly empty at the prevailing similar to 3 x 10(-2) M concentration of the membrane ubiquinone pool (Q(pool)). In each mutant, the affinity of the Q(os) domain remained typically 10-20-fold higher than that of the Q(ow) domain, as is found for wild type, thereby indicating that the single mutations caused comparable extents of the weakening at each domain. Moreover, the substitutions were found to cause similar decreases of the affinities of both Q and QH(2) in each domain, thereby maintaining the Q/QH(2) redox midpoint potentials (E(m7)) of the Q(o) site at values similar to that of the wild type. Measurement of the yield and rate of QH(2) oxidation generated by single turnover flashes in the family of mutants suggests that the Q(os) and Q(ow) domains serve different roles for the catalytic process. The yield of the QH(2) oxidation correlates linearly with Q(os) domain occupancy (QH(2) or Q), suggesting that the Q(os) domain exchanges Q or QH(2) with the Q(pool) at a rate which is much slower than the time scale of turnover. On the other hand, the rate constants of the first QH(2) oxidation, ranging in the mutants from 1620 to <5 s(-1), correlate with the K-D values of QH(2) and Q at the Q(ow) domain in a simple kinetic model in which the Q(ow) domain exchanges Q or QH(2) with the Q(pool) at a rate which is much faster than the time scale of turnover as constrained by the k(cat) (approximately 1700 s(-1)). The second QH(2) oxidation at the Q(o) site (required for completion of the catalytic turnover of the cyt bc(1) complex) proceeds maximally at 350 s(-1) in the wild type, and the yield and rate are affected by the single substitutions at F144 and G158 in parallel to those of the first QH(2) oxidation. A plausible mechanism is presented in which the two ubiquinones of the Q(o) site cooperate in the primary steps of the catalytic action of the cyt bc(1) complex. Key features of the mechanism are as follows: (1) The formation of ubisemiquinone in both the Q(os) and Q(ow) domains is highly unfavorable. This keeps the steady-state concentration of the reactive semiquinone to vanishingly low levels, and hence diminishes wasteful side reactions. (2) The Q(os) and Q(ow) domains provide a conduit for the rapid movement of semiquinone away from the oxidizing side (the [2Fe-2S] cluster, cyt c(1) and cyt c(2)) to reduce the cyt b(L). This process confers the directional specificity of the reaction, and minimizes the lifetime of semiquinone and wasteful side reactions. (3) A linear arrangement of the ubiquinones in the Q(os) and Q(ow) domains allows the position of the cyt b(L) to be at a maximum distance from the [2Fe-2S] cluster and thus stabilizes ferro-cyt b(L) with respect to the wasteful back-reaction from ferro-cyt b(L) to reoxidized [2Fe-2S] cluster. This strongly favors the physiologically useful electron transfer from ferro-cyt b(L) to ferri-cyt b(H) and the Q in the Q(i) site.