An ancient glaucophyte c6-like cytochrome related to higher plant cytochrome c6A is imported into muroplasts

Cytochrome c6 is a redox carrier in the thylakoid lumen of cyanobacteria and some eukaryotic algae. Although the isofunctional plastocyanin is present in land plants and the green alga Chlamydomonas reinhardtii, these organisms also possess a cytochrome c6-like protein designated as cytochrome c6A. Two other cytochrome c6-like groups, c6B and c6C, have been identified in cyanobacteria. In this study, we have identified a novel c6-like cytochrome PetJ2 which is encoded in the nuclear genome of Cyanophora paradoxa belonging to glaucophytes – the basal branch of the Archaeplastida. We propose that glaucophyte PetJ2 protein is related to cyanobacterial c6B and c6C cytochromes, and that cryptic green algal and land plant cytochromes c6A evolved from an ancestral archaeplastidial PetJ2 protein. In vitro import experiments with isolated muroplasts revealed that PetJ2 is imported into plastids. Although it harbors a twin-arginine motif in its thylakoid targeting peptide which is generally indicative of thylakoid import via Tat import pathway, our import experiments with isolated muroplasts and the heterologous pea thylakoid import system revealed that PetJ2 uses the Sec instead of the Tat import pathway.

Cytochrome c6A: discovery, structure and properties responsible for its low haem redox potential

Cytochrome c6A is a unique dithio-cytochrome of green algae and plants. It has a very similar core structure to that of bacterial and algal cytochromes c6, but is unable to fulfil the same function of transferring electrons from cytochrome f to Photosystem I. A key feature of cytochrome c6A is that its haem midpoint potential is more than 200 mV below that of cytochrome c6 (Em≈+340 mV) despite both cytochromes having histidine and methionine residues as axial haem-iron ligands. One salient difference between the haem pockets is that a valine residue in cytochrome c6A replaces a highly conserved glutamine residue in cytochrome c6. This difference has been probed using site-directed mutagenesis, X-ray crystallography and protein film voltammetry studies. It has been found that the stereochemistry of the glutamine residue within the haem pocket has a destabilizing effect and is responsible for tuning the haem's midpoint potential by over 100 mV. This large effect may have contributed to the evolution of a new biological function for cytochrome c6A.