Assembly and Stability of the Transmembrane Cytochrome b6

Cytochrome b₆ is a core subunit of the cytochrome b₆f complex, which is part of the photosynthetic electron transport chain. It consists of four TM helices that bind two heme cofactors non-covalently. As cytochrome b₆ serves as an excellent model for analyzing folding, assembly and stability of cofactor containing TM proteins, we analyze interactions of individual TM helices, of protein fragments as well as the contribution of cofactors and the soluble domains in vitro as well as in vivo to eventually describe the complete assembly pathway of the holo-cytochrome. After heterologous expression of the spinach cytochrome b₆ in E. coli the protein was dissolved in a mild detergent and the holo-cytochrome was reconstituted in vitro from the purified apo-protein and free heme. Mutation of histidine residues, which are crucial for heme binding, severely affected assembly and stability of the holo-cytochrome within a membrane as well as in vitro in detergents. In order to dissect the cytochrome b₆ assembly pathway in more detail, heme binding properties of isolated proteins were characterized. Based on analyzes of single and double mutants we conclude that the two hemes b_L and b_H bind in subsequent steps to the apo-protein. Binding of the heme b_L is a prerequisite for binding of heme b_H.