The High-Spin Heme bL Mutant Exposes Dominant Reaction Leading to the Formation of the Semiquinone Spin-Coupled to the [2Fe-2S]+ Cluster at the Qo Site of Rhodobacter capsulatus Cytochrome bc1

Abstract

Cytochrome bc₁ (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_LRhodobacter capsulatus cytochrome bc₁ 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 bc₁.