Phylogenetic and functional analysis of cyanobacterial Cytochrome c₆-like proteins

All known photosynthetic cyanobacteria carry a cytochrome c(6) protein that acts transferring electrons from cytochrome b(6)f complex to photosystem I, in photosynthesis, or cytochrome c oxidase, in respiration. In most of the cyanobacteria, at least one homologue to cytochrome c(6) is found, the so-called cytochrome c(6)B or cytochrome c(6)C. However, the function of these cytochrome c(6)-like proteins is still unknown. Recently, it has been proposed a common origin of these proteins as well as the reclassification of the cytochrome c(6)C group as c(6)B, renaming the new joint group as cytochrome c(6)BC. Another homologue to cytochrome c(6) has not been classified yet, the formerly called cytochrome c(6-3), which is present in the heterocyst-forming filamentous cyanobacteria Nostoc sp. PCC 7119. In this work, we propose the inclusion of this group as an independent group in the genealogy of cytochrome c(6)-like proteins with significant differences from cytochrome c(6) and cytochrome c(6)BC, with the proposed name cytochrome c(6)D. To support this proposal, new data about phylogeny, genome localisation and functional properties of cytochrome c(6)-like proteins is provided. Also, we have analysed the interaction of cytochrome c(6)-like proteins with cytochrome f by isothermal titration calorimetry and by molecular docking, concluding that c(6)-like proteins could interact with cytochrome b(6)f complex in a similar fashion as cytochrome c(6). Finally, we have analysed the reactivity of cytochrome c(6)-like proteins with membranes enriched in terminal oxidases of cyanobacteria by oxygen uptake experiments, concluding that cytochrome c(6)D is able to react with the specific copper-oxidase of the heterocysts, the cytochrome c oxidase 2.