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Heme molecules play important roles in electron transfer by redox proteins such as cytochromes. In addition, a structural role for heme in protein folding and the assembly of enzymes has been suggested. Previous results obtained using Escherichia coli hemA mutants, which are unable to synthesize 5-aminolevulinic acid, a precursor of porphyrins and hemes, have demonstrated a requirement for heme biosynthesis in the assembly of a functional succinate-ubiquinone reductase (SQR or complex II), which is a component of the aerobic respiratory chain. In the present study, in order to investigate the role of the heme in the assembly of E. coli SQR, we used a hemH (encodes ferrochelatase) mutant that lacks the ability to insert iron into the porphyrin ring. The hemH mutant failed to insert functional SQR into the cytoplasmic membrane, and the catalytic portion of SQR [the flavoprotein subunit (Fp) and the iron-sulfur protein subunit (Ip)] was localized in the cytoplasm of the cell. It is of interest to note that protoporphyrin IX accumulated in the mutant cells and inactivated the cytoplasmic succinate dehydrogenase (SDH) activity associated with the catalytic Fp-Ip complex. In contrast, SQR was assembled into the membrane of a heme-permeable hemH double mutant when hemin was present in the culture. Only a low level of SQR activity was found in the membrane when hemin was replaced by non-iron metalloporphyrins: Mn-, Co-, Ni-, Zn- and Cu-protoporphyrin IX, or protoporphyrin IX These results indicate that heme iron is indispensable for the functional assembly of SQR in the cytoplasmic membrane of E. coli, and provide a new insight into the biological role of heme in the molecular assembly of the multi-subunit enzyme complex.