Genetic fusion of subunits I, II, and 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-subunit enzyme which is a member of the superfamily of heme-copper respiratory oxidases. Three of the subunits (I, II, and III) are homologous to the three mitochondrial encoded subunits of the eukaryotic aa3-type cytochrome c oxidase. Subunits, I, II, and III of the eukaryotic oxidase 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, subunit 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 transmembrane helix at the N-terminus predicts that the amino terminal residue should be on the periplasmic side of the membrane. By deleting the intergenic region between the cyoA and cyoB genes, an in-frame fusion between subunit II (cyoA) and subunit I (cyoB) was generated. This links the C-terminus of subunit II, known to be on the periplasmic side of the membrane, to the N-terminus of subunit I. The resulting oxidase is fully active, and supports the toplogical folding pattern previously suggested for subunit I with the N-terminus in the periplasm. Whereas subunit I of the E. coli oxidase has two additional membrane-spanning helices at the C-terminus, subunit III has two fewer helices than does the corresponding subunit III of the eukaryotic oxidase.(ABSTRACT TRUNCATED AT 250 WORDS)
Ma
J
J
School of Chemical Sciences, University of Illinois, Urbana 61801.
Lemieux
L
L
Gennis
R B
RB
D001483:Base Sequence; D003576:Electron Transport Complex IV / Q000235:genetics / Q000378:metabolism*; D004789:Enzyme Activation; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D009876:Operon; D010957:Plasmids; D011993:Recombinant Fusion Proteins
D009838:Oligodeoxyribonucleotides; D011993:Recombinant Fusion Proteins; C038862:cytochrome o oxidase; D003576:Electron Transport Complex IV