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 oxidoreducta se (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, me diated by the cyt c(1) and [2Fe-2S] cluster of the cytochrome bc(1) co mplex. 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 con taining polypeptide of the Rhodobacter capsulatus cyt bc(1) complex we re 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 o f the strong and weak binding Q(o) site domains (Q(os) and Q(ow)). Var ious 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 zer o 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 empt y at the prevailing similar to 3 x 10(-2) M concentration of the membr ane ubiquinone pool (Q(pool)). In each mutant, the affinity of the Q(o s) 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. M oreover, the substitutions were found to cause similar decreases of th e affinities of both Q and QH(2) in each domain, thereby maintaining t he Q/QH(2) redox midpoint potentials (E(m7)) of the Q(o) site at value s 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 r oles for the catalytic process. The yield of the QH(2) oxidation corre lates linearly with Q(os) domain occupancy (QH(2) or Q), suggesting th at the Q(os) domain exchanges Q or QH(2) with the Q(pool) at a rate wh ich 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 mutan ts 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) doma in exchanges Q or QH(2) with the Q(pool) at a rate which is much faste r than the time scale of turnover as constrained by the k(cat) (approx imately 1700 s(-1)). The second QH(2) oxidation at the Q(o) site (requ ired 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 para llel to those of the first QH(2) oxidation. A plausible mechanism is p resented in which the two ubiquinones of the Q(o) site cooperate in th e primary steps of the catalytic action of the cyt bc(1) complex. Key features of the mechanism are as follows: (1) The formation of ubisemi quinone in both the Q(os) and Q(ow) domains is highly unfavorable. Thi s 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 moveme nt 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 th e directional specificity of the reaction, and minimizes the lifetime of semiquinone and wasteful side reactions. (3) A linear arrangement o f the ubiquinones in the Q(os) and Q(ow) domains allows the position o f the cyt b(L) to be at a maximum distance from the [2Fe-2S] cluster a nd thus stabilizes ferro-cyt b(L) with respect to the wasteful back-re action from ferro-cyt b(L) to reoxidized [2Fe-2S] cluster. This strong ly favors the physiologically useful electron transfer from ferro-cyt b(L) to ferri-cyt b(H) and the Q in the Q(i) site.