Evidence for a hetero-oligomeric structure of the chloroplast cytochrome b-559

A second nonhomologous polypeptide in the thylakoid membrane cytochrome b-559 has been suggested by the finding of a smaller reading frame just slightly downstream from that corresponding to the 9 kDa cytochrome polypeptide that is dominant on a Coomassie-stained gel. This reading frame encoded a 39-residue polypeptide that was similar in having a central hydrophobic domain of 25–26 residues and a single His residue at the same position in the hydrophobic domain. The smallest polypeptide seen on SDS gels of the cytochrome was isolated by high-performance liquid chromatography (HPLC). The NH₂-terminal sequence matched that of the downstream gene. The stoichiometry of the 2 gene products separated by HPLC was approx. 1:1, based on the molecular masses of 9.16 and 4.27 kDa calculated from the nucleotide sequence. It is concluded that the cytochrome contains both the 9.16 kDa (α) and 4.27 kDa (β) polypeptides. These data, the single His residue on each polypeptide, and the previous finding of a bis-histidine coordination, imply that the unit heme binding structure of the cytochrome is a heme cross-linked dimer. If the cytochrome contains a single heme, the dimer structure would be (αβ). If there are 2 hemes/cytochrome, the more likely structure would be (αβ)₂, a tetramer consisting of 2 heme cross-linked hetero-dimers.