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 _6B or cytochrome c _6C. 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 _6C group as c _6B, renaming the new joint group as cytochrome c _6BC. 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 _6BC, with the proposed name cytochrome c _6D. 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 _6D is able to react with the specific copper-oxidase of the heterocysts, the cytochrome c oxidase 2.