ABSTRACT
The ubiquinol-cytochrome c oxidoreductases, central to cellular respiration and photosynthesis, are homodimers. High symmetry has frustrated resolution of whether cross-dimer interactions are functionally important. This has resulted in a proliferation of contradictory models. Here, we duplicated and fused cytochrome b subunits, and then broke symmetry by introducing independent mutations into each monomer. Electrons moved freely within and between monomers, crossing an electron-transfer bridge between two hemes in the core of the dimer. This revealed an H-shaped electron-transfer system that distributes electrons between four quinone oxidation-reduction terminals at the corners of the dimer within the millisecond time scale of enzymatic turnover. Free and unregulated distribution of electrons acts like a molecular-scale bus bar, a design often exploited in electronics.
Subject(s)
Electron Transport Complex III/chemistry , Electron Transport Complex III/metabolism , Electron Transport , Electrons , Rhodobacter capsulatus/enzymology , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Cytochromes c/metabolism , Electron Transport Complex III/genetics , Models, Molecular , Mutant Proteins/chemistry , Mutant Proteins/metabolism , Oxidation-Reduction , Point Mutation , Protein Conformation , Protein Multimerization , Protein Subunits/chemistry , Protein Subunits/metabolism , Ubiquinone/analogs & derivatives , Ubiquinone/metabolismABSTRACT
In addition to its bioenergetic function of building up proton motive force, cytochrome bc1 can be a source of superoxide. One-electron reduction of oxygen is believed to occur from semiquinone (SQ(o)) formed at the quinone oxidation/reduction Q(o) site (Q(o)) as a result of single-electron oxidation of quinol by the iron-sulfur cluster (FeS) (semiforward mechanism) or single-electron reduction of quinone by heme b(L) (semireverse mechanism). It is hotly debated which mechanism plays a major role in the overall production of superoxide as experimental data supporting either reaction exist. To evaluate a contribution of each of the mechanisms we first measured superoxide production under a broad range of conditions using the mutants of cytochrome bc1 that severely impeded the oxidation of FeS by cytochrome c1, changed density of FeS around Q(o) by interfering with its movement, or combined these two effects together. We then compared the amount of generated superoxide with mathematical models describing either semiforward or semireverse mechanism framed within a scheme assuming competition between the internal reactions at Q(o) and the leakage of electrons on oxygen. We found that only the model of semireverse mechanism correctly reproduced the experimentally measured decrease in ROS for the FeS motion mutants and increase in ROS for the mutants with oxidation of FeS impaired. This strongly suggests that this mechanism dominates in setting steady-state levels of SQ(o) that present a risk of generation of superoxide by cytochrome bc1. Isolation of this reaction sequence from multiplicity of possible reactions at Q(o) helps to better understand conditions under which complex III might contribute to ROS generation in vivo.
