Abstract
Cytochrome bc (1) (mitochondrial complex III) catalyzes electron transfer from quinols to cytochrome c and couples this reaction with proton translocation across lipid membrane; thus, it contributes to the generation of protonmotive force used for the synthesis of ATP. The energetic efficiency of the enzyme relies on a bifurcation reaction taking place at the Q(o) site which upon oxidation of ubiquinol directs one electron to the Rieske 2Fe2S cluster and the other to heme b (L). The molecular mechanism of this reaction remains unclear. A semiquinone spin-coupled to the reduced 2Fe2S cluster (SQ(o)-2Fe2S) was identified as a state associated with the operation of the Q(o) site. To get insights into the mechanism of the formation of this state, we first constructed a mutant in which one of the histidine ligands of the iron ion of heme b (L) Rhodobacter capsulatus cytochrome bc (1) was replaced by asparagine (H198N). This converted the low-spin, low-potential heme into the high-spin, high-potential species which is unable to support enzymatic turnover. We performed a comparative analysis of redox titrations of antimycin-supplemented bacterial photosynthetic membranes containing native enzyme and the mutant. The titrations revealed that H198N failed to generate detectable amounts of SQ(o)-2Fe2S under neither equilibrium (in dark) nor nonequilibrium (in light), whereas the native enzyme generated clearly detectable SQ(o)-2Fe2S in light. This provided further support for the mechanism in which the back electron transfer from heme b (L) to a ubiquinone bound at the Q(o) site is mainly responsible for the formation of semiquinone trapped in the SQ(o)-2Fe2S state in R. capusulatus cytochrome bc (1).