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 b_L and b_H bind in two subsequent steps to the apo-protein. Binding of the low-potential heme b_L is a prerequisite for binding the high-potential heme b_H. After substitution of His86, which serves as an axial ligand for heme b_L, the apo-protein did not bind heme, while substitution of the heme b_L-ligating residue His187 still allowed binding of both hemes. Similarly, after replacement of His202, one axial ligand to heme b_H, binding of only heme b_L was observed, whereas replacement of His100, the other heme b_H 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 b_L or heme b_H), 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.