Soluble variants of Rhodobacter capsulatus membrane-anchored cytochrome c_y are efficient photosynthetic electron carriers

Photosynthetic (Ps) electron transport pathways often contain multiple electron carriers with overlapping functions. Here we focus on two c-type cytochromes (cyt) in facultative phototrophic bacteria of the Rhodobacter genus: the diffusible cyt c₂ and the membrane-anchored cyt c_y. In species like R. capsulatus, cyt c_y functions in both Ps and respiratory electron transport chains, whereas in other species like R. sphaeroides, it does so only in respiration. The molecular bases of this difference was investigated by producing a soluble variant of cyt c_y (S-c_y), by fusing genetically the cyt c₂ signal sequence to the cyt c domain of cyt c_y. This novel electron carrier was unable to support the Ps growth of R. capsulatus. However, strains harboring cyt S-c_y regained Ps growth ability by acquiring mutations in its cyt c domain. They produced cyt S-c_y variants at amounts comparable with that of cyt c₂, and conferred Ps growth. Chemical titration indicated that the redox midpoint potential of cyt S-c_y was about 340 mV, similar to that of cyts c₂ or c_y. Remarkably, electron transfer kinetics from the cyt bc₁ complex to the photochemical reaction center (RC) mediated by cyt S-c_y was distinct from those seen with the cyt c₂ or cyt c_y. The kinetics exhibited a pronounced slow phase, suggesting that cyt S-c_y interacted with the RC less tightly than cyt c₂. Comparison of structural models of cyts c₂ and S-c_y revealed that several of the amino acid residues implicated in long-range electrostatic interactions promoting binding of cyt c₂ to the RC are not conserved in cyt c_y, whereas those supporting short-range hydrophobic interactions are conserved. These findings indicated that attaching electron carrier cytochromes to the membrane allowed them to weaken their interactions with their partners so that they could accommodate more rapid multiple turnovers.