Modulation of heme redox potential in the cytochrome c6 family

Cytochrome c(6A) is a unique dithio-cytochrome of green algae and plants. It has a very similar core structure to that of bacterial and algal cytochromes c(6) but is unable to fulfill the same function of transferring electrons from cytochrome f to photosystem I. A key feature is that its heme midpoint potential is more than 200 mV below that of cytochrome c(6) despite having His and Met as axial heme-iron ligands. To identify the molecular origins of the difference in potential, the structure of cytochrome c(6) from the cyanobacterium Phormidium laminosum has been determined by X-ray crystallography and compared with the known structure of cytochrome c(6A). One salient difference of the heme pockets is that a highly conserved Gln (Q51) in cytochrome c(6) is replaced by Val (V52) in c(6A). Using protein film voltammetry, we found that swapping these residues raised the c(6A) potential by +109 mV and decreased that of c(6) by almost the same extent, -100 mV. X-ray crystallography of the V52Q protein showed that the Gln residue adopts the same configuration relative to the heme as in cytochrome c(6) and we propose that this stereochemistry destabilizes the oxidized form of the heme. Consequently, replacement of Gln by Val was probably a key step in the evolution of cytochrome c(6A) from cytochrome c(6), inhibiting reduction by the cytochrome b(6)f complex and facilitating establishment of a new function.