Heme incorporation into the cytochrome bo3 occurs at a late stage of assembly.

Respiratory complexes in both prokaryotes and eukaryotes contain multiple co-factors, which are coordinated in defined positions so that they can function as electron wires. Intriguingly, co-factors are usually buried deep within hetero-oligomeric protein complexes and it is not clear when or how they are incorporated. In this study we show that heme is incorporated into the cytochrome bo(3) complex of Escherichia coli at a late stage of assembly. Specifically the apo-form of subunit I (the catalytic subunit) interacts with subunits III and IV before accepting heme. Assembly of subunit II is stalled until heme is incorporated.