Exploring subunit-subunit interactions in the Escherichia coli bo-type ubiquinol oxidase by extragenic suppressor mutation analysis.
Cytochrome bo-type ubiquinol oxidase is a four-subunit heme-copper terminal oxidase and functions as a redox-coupled proton pump in the aerobic respiratory chain of Escherichia coli. On the basis of deletion and chemical cross-linking analyses on subunit IV, we proposed that subunit IV is essential for CuB binding to subunit I and that it is present in a cleft between subunits I and III (Saiki, K., Nakamura, H., Mogi, T., and Anraku, Y. (1996) J. Biol. Chem. 271, 15336-15340). To extend previous studies, we carried out alanine-scanning mutagenesis for selected 16-amino acid residues in subunit IV to explore subunit-subunit interactions in bo-type ubiquinol oxidase. We found that only the replacement of Phe83 in helix III resulted in the reduction of the catalytic activity but that this did not significantly affect the UV-visible spectroscopic properties and the copper content. This suggests that individual amino acid substitutions, including the six invariant residues, are not enough to alter such properties of the metal centers. Extragenic suppressor mutations were isolated for the Phe83 --> Ala mutation of subunit IV and identified as missense mutations in helices VII and VIII in subunit I. These observations provide further support for specific interactions of subunit IV with helix VII and/or VIII, the CuB binding domain, of subunit I and suggest that subunit IV functions as a domain-specific molecular chaperon in the oxidase complex.
Saiki
K
K
Department of Biological Sciences, Graduate School of Science, University of Tokyo, Hongo, Bunkyo-ku, Tokyo 113, Japan.
Mogi
T
T
Tsubaki
M
M
Hori
H
H
Anraku
Y
Y
D000409:Alanine; D000595:Amino Acid Sequence; D001665:Binding Sites; D002462:Cell Membrane / Q000201:enzymology / Q000648:ultrastructure; D003576:Electron Transport Complex IV / Q000737:chemistry* / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics*; D007700:Kinetics; D046911:Macromolecular Substances; D008961:Models, Structural; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010649:Phenylalanine; D017354:Point Mutation; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D015183:Restriction Mapping; D013053:Spectrophotometry; D013489:Suppression, Genetic
D046911:Macromolecular Substances; D011994:Recombinant Proteins; D010649:Phenylalanine; C038862:cytochrome o oxidase; D003576:Electron Transport Complex IV; D000409:Alanine