To define the structural basis for cofactor binding to membrane proteins, we introduce a manageable model system, which allows us, for the first time, to study the influence of individual transmembrane helices and of single amino acid residues on the assembly of a transmembrane cytochrome. In vivo as well as in vitro analyses indicate central roles of single amino acid residues for either interaction of the transmembrane helices or for binding of the cofactor. The results clearly show that interaction of the PsbF transmembrane helix is independent from binding of the heme cofactor. On the other hand, binding of the cofactor highly depends on helix-helix interactions. By site-directed mutagenesis critical amino acid residues were identified, which are involved in the assembly of a functional transmembrane cytochrome. Especially, a highly conserved glycine residue is critical for interaction of the transmembrane helices and assembly of the cytochrome. Based on the two-stage-model of alpha-helical membrane protein folding, the presented results clearly indicate a third stage of membrane protein folding, in which a cofactor binds to a pre-assembled transmembrane protein.