Intermonomer Electron Transfer between the b Hemes of Heterodimeric Cytochrome bc1

The ubihydroquinone:cytochrome c oxidoreductase, or cytochrome bc(1), is a central component of respiratory and photosynthetic energy transduction pathways in many organisms. It contributes to the generation of membrane potential and proton gradient used for cellular energy (ATP) production. The three-dimensional structures of cytochrome bc(1) show a homodimeric organization of its three catalytic subunits. The unusual architecture revived the issue of whether the monomers operate independently or function cooperatively during the catalytic cycle of the enzyme. In recent years, different genetic approaches allowed the successful production of heterodimeric cytochrome bc(1), variants and evidenced the occurrence of intermonomer electron transfer between the monomers of this enzyme. Here we used a version of the two-plasmid genetic system, also described in the preceding paper (DOI: 10.1021/bi400560p), to study a new heterodimeric mutant variant of cytochrome bc(1). The strain producing this heterodimeric variant sustained photosynthetic growth of Rhodobacter capsulatus and yielded an active heterodimer. Interestingly, kinetic data showed equilibration of electrons among the four b heme cofactors of the heterodimer, via reverse intermonomer electron transfer between the b(L) hemes. Both inactive homodimeric and active heterodimeric cytochrome bc(1), variants were purified to homogeneity from the same cells, and purified samples were subjected to mass spectrometry analyses. The data unequivocally supported the idea that the cytochrome b subunits carried the expected mutations and their associated epitope tags. Implications of these findings on our interpretation of light-activated transient cytochrome b and c redox kinetics and the mechanism of function of a dimeric cytochrome bc(1), are discussed with respect to the previously proposed heterodimeric Q cycle model.