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 ter minal oxidase and functions as a redox-coupled proton pump in the aero bic respiratory chain of Escherichia cell. On the basis of deletion an d chemical cross-linking analyses on subunit IV, we proposed that subu nit IV is essential for Cu-B binding to subunit I and that it is prese nt in a cleft between subunits I and III (Saiki, K., Nakamura, H., Mog i, T., and Anraku, Y. (1996) J. Biol. Chem. 271, 15336-15340). To exte nd previous studies, we carried out alanine-scanning mutagenesis for s elected 16-amino acid residues in subunit IV to explore subunit-subuni t interac tions in bo-type ubiquinol oxidase. We found that only the r eplacement of Phe(83) in helix III resulted in the reduction of the ca talytic activity but that this did not significantly affect the UV-vis ible spectroscopic properties and the copper content. This suggests th at individual amino acid substitutions, including the six invariant re sidues, are not enough to alter such properties of the metal centers. Extragenic suppressor mutations were isolated for the Phe(83) --> 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, t he Cu-B binding domain, of subunit I and suggest that subunit IV funct ions as a domain-specific molecular chaperon in the oxidase complex.