In vivo and in vitro requirements for the formation of cytochrome b 6 were examined to analyze the mechanisms of transmembrane b-type cytochrome formation. After heterologous expression of spinach cytochrome b 6 , formation of the holo-cytochrome was observed within the E. coli inner membrane. The transmembrane orientation of cytochrome b 6 appeared not to be critical for heme binding and holo-cytochrome formation. Furthermore, in vitro reconstitution of cytochrome b 6 was possible under oxidizing as well as under reducing conditions. Taken together these observations strongly indicate that transmembrane b-type cytochromes can spontaneously assemble in vitro as well as in a membrane.

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