Structural model of cytochrome b₅₅₉ in photosystem II based on a mutant with genetically fused subunits

Photosystem II is a reaction center protein complex located in photosynthetic membranes of plants, algae, and cyanobacteria. Using light energy, photosystem II catalyzes the oxidation of water and the reduction of plastoquinone, resulting in the release of molecular oxygen. A key component of photosystem II is cytochrome b₅₅₉, a membrane-embedded heme protein with an unknown function. The cytochrome is unusual in that a heme links two separate polypeptide subunits, α and β, either as a heterodimer (αβ) or as two homodimers (α₂ and β₂). To determine the structural organization of cytochrome b₅₅₉ in the membrane, we used site-directed mutagenesis to fuse the coding regions of the two respective genes in the cyanobacterium Synechocystis sp. PCC 6803. In this construction, the C terminus of the α subunit (9 kDa) is attached to the N terminus of the β subunit (5 kDa) to form a 14-kDa αβ fusion protein that is predicted to have two membrane-spanning α-helices with antiparallel orientations. Cells containing the αβ fusion protein grow photoautotrophically and assemble functional photosystem II complexes. Optical spectroscopy shows that the αβ fusion protein binds heme and is incorporated into photosystem II. These data support a structural model of cytochrome b₅₅₉ in which one heme is coordinated to an α₂ homodimer and a second heme is coordinated to a β₂ homodimer. In this model, each photosystem II complex contains two cytochrome b₅₅₉ hemes, with the α₂ heme located near the stromal side of the membrane and the β₂ heme located near the lumenal side.