Substitutions of conserved aromatic amino acid residues in subunit I perturb the metal centers of the Escherichia coli bo-type ubiquinol oxidase

摘要

Cytochrome bo is a four-subunit quinol oxidase in the aerobic respiratory chain of Escherichia coli and functions as a redox-coupled proton pump. Subunit I binds all the redox metal centers, low-spin heme b, high-spin heme o, and Cu-B, whose axial ligands have been identified to be six invariant histidines. This work explored the possible roles of the aromatic amino acid residues conserved in the putative transmembrane helices (or st the boundary of the membrane) of subunit I. Sixteen aromatic amino acid residues were individually substituted by Leu, except for Tyr(61) and Trp(282) by Phe and Phe(415) by Trp, Leu substitutions of Trp(280) and Tyr(288) in helix VI, Trp(331) in loop VII-VIII, and Phe(348) in helix VIII reduced the catalytic activity, whereas all other mutations did not affect the in vivo activity. Spectroscopic analyses of the purified mutant enzymes revealed that the defects were attributable to perturbations of the binuclear center. On the basis of these findings and recent crystallographic studies on cytochrome c oxidases, we discuss the possible roles of the conserved aromatic amino acid residues in subunit I of the heme-copper terminal oxidases. [References: 55]