The first atomic structure of a subunit of either the cytochrome b6f complex or of the related cytochrome bc1 complex has been obtained. The structure of cytochrome f allows prediction of the approximate docking site of plastocyanin and should allow systematic studies of the mechanism of intra- and inter-protein electron transfer between the cytochrome heme and plastocyanin copper, which are approximately isopotential. The unprecedented axial heme iron ligand also provides information on the sequence of events (i.e. cleavage of signal peptide and ligation of heme) associated with translocation of the cytochrome across the membrane and its subsequent folding.

The cyanobacterium Synechococcus sp. PCC 7002 carries two genes, petJ1 and petJ2, for proteins related to soluble, cytochrome c6 electron transfer proteins. PetJ1 was purified from the cyanobacterium, and both cytochromes were expressed with heme incorporation in Escherichia coli. The expressed PetJ1 displayed spectral and biochemical properties virtually identical to those of PetJ1 from Synechococcus. PetJ1 is a typical cytochrome c6 but contains an unusual KDGSKSL insertion. PetJ2 isolated from E. coli exhibited absorbance spectra characteristic of cytochromes, although the alpha, beta, and gamma bands were red-shifted relative to those of PetJ1. Moreover, the surface electrostatic properties and redox midpoint potential of PetJ2 (pI 9.7; E(m,7) = 148 +/- 1.7 mV) differed substantially from those of PetJ1 (pI 3.8; E(m,7) = 319 +/- 1.6 mV). These data indicate that the PetJ2 cytochrome could not effectively replace PetJ1 as an electron acceptor for the cytochrome bf complex in photosynthesis. Phylogenetic comparisons against plant, algal, bacterial, and cyanobacterial genomes revealed two novel and widely distributed clusters of previously uncharacterized, cyanobacterial c 6-like cytochromes. PetJ2 belongs to a group that is distinct from both c6 cytochromes and the enigmatic chloroplast c 6A cytochromes. We tentatively designate the PetJ2 group as c6C cytochromes and the other new group as c6B cytochromes. Possible functions of these cytochromes are discussed.

Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) biosynthesis is a multi-step process in which specific chaperones are involved. Recently, a novel polypeptide, Rubisco Accumulation Factor 1 (RAF1), has been identified as a protein that is necessary for proper assembly of this enzyme in maize cells (Zea mays). However, neither its specific function nor its mode of action have as yet been determined. The results presented here show that the prokaryotic homolog of RAF1 from Thermosynechococcus elongatus is expressed in cyanobacterial cells and interacts with a large Rubisco subunit (RbcL). Using a heterologous expression system, it was demonstrated that this protein promotes Rubisco assembly in Escherichia coli cells. Moreover, when co-expressed with RbcL alone, a stable RbcL-RAF1 complex is formed. Molecular mass determination for this Rubisco assembly intermediate by size-exclusion chromatography coupled with multiangle light scattering indicates that it consists of an RbcL dimer and two RAF1 molecules. A purified RbcL-RAF1 complex dissociated upon addition of a small Rubisco subunit (RbcS), leading to formation of the active holoenzyme. Moreover, titration of the octameric (RbcL 8 ) core of Rubisco with RAF1 results in disassembly of such a stucture and creation of an RbcL-RAF1 intermediate. The results presented here are the first attempt to elucidate the role of cyanobacterial Rubisco Accumulation Factor 1 in the Rubisco biosynthesis process. Structured digital abstract• RAF1 physically interacts with RbcL by pull down (View interaction) • RAF1 and RbcL bind by molecular sieving (View interaction)