A Robust Genetic System for Producing Heterodimeric Native and Mutant Cytochrome bc₁

Abstract

The ubihydroquinone:cytochrome c oxidoreductase, or cytochrome bc1, is central to the production of ATP by oxidative phosphorylation and photophosphorylation in many organisms. Its three-dimensional structure depicts it as a homodimer with each monomer composed of the Fe-S protein, cytochrome b, and cytochrome c1 subunits. Recent genetic approaches successfully produced heterodimeric variants of this enzyme, providing insights into its mechanism of function. However, these experimental setups are inherently prone to genetic rearrangements as they carry repeated copies of cytochrome bc1 structural genes. Duplications present on a single replicon (one-plasmid system) or a double replicon (two-plasmid system) could yield heterogeneous populations via homologous recombination or other genetic events at different frequencies, especially under selective growth conditions. In this work, we assessed the origins and frequencies of genetic variations encountered in these systems and describe an improved variant of the two-plasmid system. We found that use of a recombination-deficient background (recA) minimizes spontaneous formation of co-integrant plasmids and renders the homologous recombination within the cytochrome b gene copies inconsequential. On the basis of the data, we conclude that both the newly improved RecA-deficient and the previously used RecA-proficient two-plasmid systems reliably produce native and mutant heterodimeric cytochrome bc1 variants. The two-plasmid system developed here might contribute to the study of “mitochondrial heteroplasmy”-like heterogeneous states in model bacteria (e.g., Rhodobacter species) suitable for bioenergetics studies. In the following paper (DOI 10.1021/bi400561e), we describe the use of the two-plasmid system to produce and characterize, in membranes and in purified states, an active heterodimeric cytochrome bc1 variant with unusual intermonomer electron transfer properties.

Catalytic Reactions and Energy Conservation in the Cytochrome bc1 and b6f Complexes of Energy-Transducing Membranes

“…A second approach for obtaining asymmetrically mutated variants of Cyt bc 1 was based on coexpression of two plasmids, each carrying one copy of the gene encoding for Cyt b with a sequence coding for a different tag attached (His-tag, Flag-tag or Strep-tag). ,, The different mutations inactivating either the Q o or the Q i sites were placed on different plasmids and the two-step affinity chromatography was used to isolate the cross-inactivated form from the symmetric variants resulting from random assembly of the complexes in the cells. , These studies also demonstrated that intermonomer electron transfer through b L hemes occurs in milliseconds, thus in a catalytically relevant time scale. In addition, some of the kinetic transients were interpreted as resulting from electron equilibration between the two Q i sites and the four hemes b in the dimer .…”

Electronic Connection Between the Quinone and CytochromecRedox Pools and Its Role in Regulation of Mitochondrial Electron Transport and Redox Signaling

“…For cytochrome bc 1 , this concerns cytochrome b subunit which is coded by a gene located in mtDNA. Given that this gene does not differentiate between monomers and that mtDNA is present in several copies in mitochondria, the presence of mutations in just a part of the copies of the gene is likely to result in expression of both symmetrically and asymmetrically mutated dimers of cytochrome bc 1 (FIGURE 9) as observed in bacterial two-plasmid model systems (45,141,151). Thus it can be anticipated that the amount of the asymmetrically damaged complexes increases with age.…”

Hybrid fusions show that inter-monomer electron transfer robustly supports cytochrome bc1 function in vivo

“…It is clear that both systems (i.e., the system based on fusion protein used in this work and the two-plasmid system) consistently indicate that cytochrome bc 1 complexes modified specifically so that the only route connecting the Q o site with the Q i site involves electron transfer between the two hemes b L , are enzymatically active and efficient to sustain the cytochrome bc 1 -dependent photoheterotrophic growth of the cells. This further rules out the arguments against the existence of inter-monomer electron transfer [17] (detailed discussion dealing with these arguments can be found in [15,26] ).…”