Multiple Step Assembly Of The Transmembrane Cytochrome b6

We have analyzed the role of individual heme-ligating histidine residues for assembly of holo-cytochrome b6, and we show that the two hemes bL and bH bind in two subsequent steps to the apo-protein. Binding of the low-potential heme bL is a prerequisite for binding the high-potential heme bH. After substitution of His86, which serves as an axial ligand for heme bL, the apo-protein did not bind heme, while substitution of the heme bL-ligating residue His187 still allowed binding of both hemes. Similarly, after replacement of His202, one axial ligand to heme bH, binding of only heme bL was observed, whereas replacement of His100, the other heme bH ligand, resulted in binding of both hemes. These data indicate sequential heme binding during formation of the holo-cytochrome, and the two histidine residues, which serve as axial ligands to the same heme molecule (heme bL or heme bH), have different importance during heme binding and cytochrome assembly. Furthermore, determination of the heme midpoint potentials of the various cytochrome b6 variants indicates a cooperative adjustment of the heme midpoint potentials in cytochrome b6.