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

► Respiratory complexes contain multiple co-factors. ► It is not clear when or how co-factors are incorporated. ► For the cytochrome bo3 we show that heme is inserted at a late stage of assembly. ► The downstream assembly step is stalled until heme is inserted. ► The data provide a framework for assembly of heme-copper respiratory oxidases.