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

Jixiang Ma, Laura Lemieux, Robert B. Gennis

Research output: Contribution to journalArticlepeer-review

Abstract

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. Since the 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 subunits I and III occur at different points. If this is true, it should be 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 subunit III are both predicted to be on the cytoplasmic side of the membrane. By deleting the cyoB-cyoC intergenic region, the fusion of subunits 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 oxidase. The fused subunit (II-I-III) contains 22 transmembrane spans. These data support the previously proposed topology of the subunits and provide a starting point for defining how the three subunits interact with each other.

Original languageEnglish (US)
Pages (from-to)7692-7697
Number of pages6
JournalBiochemistry
Volume32
Issue number30
DOIs
StatePublished - 1993

ASJC Scopus subject areas

  • Biochemistry

Fingerprint

Dive into the research topics of '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'. Together they form a unique fingerprint.

Cite this