Generation of semiquinone-[2Fe-2S]+ spin-coupled center at the Q0 site of cytochrome bc1 in redox-poised, illuminated photosynthetic membranes from Rhodobacter capsulatus

One of the less understood parts of the catalytic cycle of cytochrome bc(1)/b(6)f complexes is the mechanism of electronic bifurcation occurring within the hydroquinone oxidation site (Q(o) site). Several models describing this mechanism invoke a phenomenon of formation of an unstable semiquinone. Recent studies with isolated cytochrome bc(1) or b(6)f revealed that a relatively stable semiquinone spin-coupled to the reduced Rieske cluster (SQ-FeS) is generated at the Q(o) site during the oxidation of ubi- or plastohydroquinone analogs under conditions of continuous turnover. Here, we identified the EPR transition of SQ-FeS formed upon oxidation of ubihydroquinone in native photosynthetic membranes from purple bacterium Rhodobacter capsulatus. We observed a significant amount of SQ-FeS generated when the antimycin-inhibited enzyme experiences conditions of non equilibrium caused by the continuous light activation of the reaction center. We also noted that SQ-FeS cannot be detected under equilibrium redox titrations in dark. The non-equilibrium redox titrations of SQ-FeS indicate that this center has a higher apparent redox midpoint potential when compared to the redox midpoint potential of the quinone pool. This suggests that SQ-FeS is stabilized, which corroborates a recently proposed mechanism in which the SQ-FeS state is metastable and functions to safely hold electrons at the local energy minimum during the oxidation of ubihydroquinone and limits superoxide formation. Our results open new possibilities to study the formation and properties of this state in cytochromes bc under close to physiological conditions in which non-equilibrium is attained by the light activation of bacterial reaction centers or photosystems.