Cytochrome (cyt) bc1 is a structural dimer with its monomers consisting of the Fe/S protein, cyt b and cyt c1 subunits. Its three-dimensional architecture depicts it as a symmetrical homo-dimer, but the mobility of the head domain of the Fe/S protein indicates that the functional enzyme undergoes asymmetrical hetero-dimeric conformations. Here, we report a new genetic system for studying intra and inter-monomer interactions within the cyt bc1 using the facultative phototrophic bacterium Rhodobacter capsulatus. The system involves two different sets of independently expressed cyt bc1 structural genes carried by two plasmids that are co-harbored by a cell without its endogenous enzyme. Our results indicate that co-expressed cyt bc1 subunits are matured, assorted and assembled in vivo into homo- and hetero-dimeric enzymes that can bear different mutations in each monomer. Using the system, the occurrence of inter-monomer electron transfer between the low potential b hemes of cyt bc1 was probed by choosing mutations that perturb electron transfer at the hydroquinone oxidation (Qo) and quinone reduction (Qi) sites of the enzyme. The data demonstrate that active hetero-dimeric variants, formed of monomers carrying mutations that abolish only one of the two (Qo or Qi) active sites of each monomer, are produced and they support photosynthetic growth of R. capsulatus. Detailed analyses of the physicochemical properties of membranes of these mutants, as well as purified homo- and hetero-dimeric cyt bc1 preparations, demonstrate that efficient and productive electron transfer occurs between the low potential hemes bL of the monomers in a hetero-dimeric enzyme. Overall findings are discussed with respect to intra- and inter-monomer interactions that occur during the catalytic turnover of cyt bc1.
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