GENETIC FUSION OF SUBUNIT-I, SUBUNIT-II, AND SUBUNIT-III OF THE CYTOCHROME-BO UBIQUINOL OXIDASE FROM ESCHERICHIA-COLI RESULTS IN A FULLY ASSEMBLED AND ACTIVE ENZYME

The cytochrome bo ubiquinol oxidase from Escherichia coli is a five-su bunit enzyme which is a member of the superfamily of heme-copper respi ratory oxidases. Three of the subunits (I, II, and III) are homologous to the three mitochondrial encoded subunits of the eukaryotic aa3-typ e cytochrome c oxidase. Subunits, I, II, and III of the eukaryotic oxi dase contain 12, 2, and 7 putative transmembrane spans, respectively. The hydropathy profiles of the subunits of most other members of this oxidase superfamily are consistent with these structures. However, sub unit I from the E. coli oxidase contains 15 transmembrane spans, with one additional span at the N-terminus and two additional spans at the C-terminus in comparison to the eukaryotic oxidase. The additional tra nsmembrane helix at the N-terminus predicts that the amino terminal re sidue should be on the periplasmic side of the membrane. By deleting t he intergenic region between the cyoA and cyoB genes, an in-frame fusi on between subunit II (cyoA) and subunit I (cyoB) was generated. This links the C-terminus of subunit II, known to be on the periplasmic sid e of the membrane, to the N-terminus of subunit I. The resulting oxida se is fully active, and supports the toplogical folding pattern previo usly suggested for subunit I with the N-terminus in the periplasm. Whe reas subunit I of the E. coli oxidase has two additional membrane-span ning helices at the C-terminus, subunit III has two fewer helices than does the corresponding subunit III of the eukaryotic oxidase. Since t he genes encoding subunits I (cyoB) and III (cyoC) are contiguous, it is tempting to speculate that the overall structures of subunits I and III are similar in the two oxidases, but that the breaks between subu nits I and III occur at different points. If this is true, it should b e possible, in principle, to fuse subunits I and III to form a single subunit (I-III). The C-terminus of subunit I and the N-terminus of sub unit III are both predicted to be on the cytoplasmic side of the membr ane. By deleting the cyoB-cyoC intergenic region, the fusion of subuni ts I and III was accomplished, and resulted in an active oxidase. When in-frame fusions were made between all three subunits (II-I-III), the resulting gene product still assembles as part of a functional oxidas e. The fused subunit (II-I-III) contains 22 transmembrane spans. These data support the previously proposed topology of the subunits and pro vide a starting point for defining how the three subunits interact wit h each other.