10.1021/bi400561e.s001 Pascal Lanciano Pascal Lanciano Bahia Khalfaoui-Hassani Bahia Khalfaoui-Hassani Nur Selamoglu Nur Selamoglu Fevzi Daldal Fevzi Daldal Intermonomer Electron Transfer between the <i>b</i> Hemes of Heterodimeric Cytochrome <i>bc</i><sub>1</sub> American Chemical Society 2016 bL hemes Rhodobacter capsulatus intermonomer electron transfer heterodimeric Q cycle model Intermonomer Electron Transfer b Hemes epitope tags photosynthetic energy transduction pathways photosynthetic growth DOI ATP b heme cofactors mass spectrometry analyses dimeric cytochrome bc 1 heterodimeric cytochrome bc 1 variants cytochrome bc 1. c redox kinetics proton gradient heterodimeric variant homodimeric organization cytochrome bc 1 show cytochrome b cytochrome b subunits 2016-02-18 15:38:40 Journal contribution https://acs.figshare.com/articles/journal_contribution/Intermonomer_Electron_Transfer_between_the_i_b_i_Hemes_of_Heterodimeric_Cytochrome_i_bc_i_sub_1_sub_/2366386 The ubihydroquinone:cytochrome <i>c</i> oxidoreductase, or cytochrome <i>bc</i><sub>1</sub>, 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 <i>bc</i><sub>1</sub> 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 <i>bc</i><sub>1</sub> 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 <i>bc</i><sub>1</sub>. The strain producing this heterodimeric variant sustained photosynthetic growth of <i>Rhodobacter capsulatus</i> and yielded an active heterodimer. Interestingly, kinetic data showed equilibration of electrons among the four <i>b</i> heme cofactors of the heterodimer, via “reverse” intermonomer electron transfer between the <i>b</i><sub>L</sub> hemes. Both inactive homodimeric and active heterodimeric cytochrome <i>bc</i><sub>1</sub> 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 <i>b</i> subunits carried the expected mutations and their associated epitope tags. Implications of these findings on our interpretation of light-activated transient cytochrome <i>b</i> and <i>c</i> redox kinetics and the mechanism of function of a dimeric cytochrome <i>bc</i><sub>1</sub> are discussed with respect to the previously proposed heterodimeric Q cycle model.