Probing a role of subunit IV of the Escherichia coli bo-type ubiquinol oxidase by deletion and cross-linking analyses

Abstract

Subunit IV of the Escherichia coli bo-type ubiquinol oxidase is a 12- kDa membrane protein encoded by the cyoD gene and is conserved in the bacterial heme-copper terminal oxidases. To probe the functional role of subunit IV, we carried out deletion analysis and chemical cross-linking experiments with a homobifunctional and clearable reagent. Spectroscopic properties of the mutant oxidases suggest that the C-terminal two-third (Val45 to His109) containing helices II and III is essential for the functional expression of the oxidase complex and for the Cu(B) binding to the heme-copper binuclear center in subunit I. Cross-linking studies indicate that subunit IV is in close vicinity to subunit III. Based on these observations, we propose that subunit IV is present in a cleft formed by subunits I and III and assists the Cu(B) binding to subunit I during biosynthesis or assembly of the oxidase complex.