Ubiquinone Pair in the Qo Site Central to the Primary Energy Conversion Reactions of Cytochrome bc1 Complex

Abstract

The mechanistic heart of the ubihydroquinone-cytochrome c oxidoreductase (cyt bc1 complex) is the catalytic oxidation of ubihydroquinone (QH2) at the Qo site. QH2 oxidation is initiated by ferri-cyt c, mediated by the cyt c1 and [2Fe-2S] cluster of the cytochrome bc1 complex. QH2 oxidation in turn drives transmembrane electronic charge separation through two b-type hemes to another ubiquinone (Q) at the Qi site. In earlier studies, residues F144 and G158 of the b-heme containing polypeptide of the Rhodobacter capsulatus cyt bc1 complex were shown to be influential in Qo 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 QH2 at each of the strong and weak binding Qo site domains (Qos and Qow). Various substitutions at F144 or G158 were found to weaken the affinities for Q and QH2 at both the Qos and Qow domains variably from zero to beyond 10(3)-fold. This produced a family of Qo sites with Qos and Qow domain occupancies ranging from nearly full to nearly empty at the prevailing approximately 3 x 10(-2) M concentration of the membrane ubiquinone pool (Qpool). In each mutant, the affinity of the Qos domain remained typically 10-20-fold higher than that of the Qow 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 QH2 in each domain, thereby maintaining the Q/QH2 redox midpoint potentials (Em7) of the Qo site at values similar to that of the wild type. Measurement of the yield and rate of QH2 oxidation generated by single turnover flashes in the family of mutants suggests that the Qos and Qow domains serve different roles for the catalytic process. The yield of the QH2 oxidation correlates linearly with Qos domain occupancy (QH2 or Q), suggesting that the Qos domain exchanges Q or QH2 with the Qpool at a rate which is much slower than the time scale of turnover.(ABSTRACT TRUNCATED AT 400 WORDS)

Mechanism of Ubiquinol Oxidation by the bc1 Complex: Different Domains of the Quinol Binding Pocket and Their Role in the Mechanism and Binding of Inhibitors

“…Since the residues contributing to these functions are not a common set, mutations might be expected at the different interfaces, at either of which changes could modify the function of the site. The extensive work from Ding et al (34)(35)(36)(37) on occupancy of the site in different mutant strains as reflected in the g x ) 1.80 signal of the [2Fe-2S] center has contributed a valuable set of data on interactions of the occupant with the ISP. In collaboration with Ding and Dutton, we have made similar measurements on some of our mutant strains from Rb.…”

“…(6) The remaining evidence for double occupancy comes from interpretation of the change in g x line shape when quinone is extracted from chromatophores (34,35). Paradoxically, as noted above, other properties attributed to the Q os and Q ow species were similar.…”

“…In the absence of inhibitors, the spectrum reflects the state of the quinone pool (31)(32)(33)(34)(35)(36)(37). When the Q pool is oxidized, a signal at the g x ) 1.800 is observed.…”

“…Further information on the occupancy of the Q o site has been obtained though study of the interaction of the occupant with the [2Fe-2S] center of the reduced ISP, observed through changes in the EPR spectrum. In addition to the effects observed with stigmatellin and UHDBT (23)(24)(25)(26), a well-defined spectral change giving rise to a band at g x ) 1.80 has been characterized when quinone and the reduced ISP are both present (31)(32)(33)(34)(35)(36)(37). The dependence on ambient redox potential, and on the extent of extraction of quinone, has lead Ding and colleagues to suggest a double occupancy of the site by weakly (Q ow ) and strongly (Q os ) binding quinone species (34)(35)(36)(37).…”

“…The dependence on ambient redox potential, and on the extent of extraction of quinone, has lead Ding and colleagues to suggest a double occupancy of the site by weakly (Q ow ) and strongly (Q os ) binding quinone species (34)(35)(36)(37). Inhibitors of either class eliminate EPR signals ascribed to both species (34,35). Support for occupancy of the Q o site by more than one species has been suggested by Brandt et al (30) on the basis of kinetic studies interpreted as showing noncompetitive binding of MOA-type inhibitors in the presence of substrate quinol.…”