SITE-DIRECTED MUTAGENESIS OF RESIDUES WITHIN HELIX-VI IN SUBUNIT-I OFTHE CYTOCHROME-BO(3) UBIQUINOL OXIDASE FROM ESCHERICHIA-COLI SUGGESTSTHAT TYROSINE-288 MAY BE A CU-B LIGAND

The heme-copper oxidase superfamily contains all of the mammalian mito chondrial cytochrome c oxidases, as well as most prokaryotic respirato ry oxidases. All members of the superfamily have a subunit homologous to subunit I of the mammalian cytochrome c oxidases. This subunit prov ides the amino acid ligands to a low-spin heme component as well as to a heme-copper binuclear center, which is the site where dioxygen is r educed to water. The amino acid sequence of transmembrane helix VI of subunit I is the most highly conserved within the superfamily. Previou s efforts have demonstrated that one of the residues in this region, H 284, is critical for oxidase activity and for the assembly of Cu-B. Th is paper presents the analysis of additional site-directed mutants in which other highly conserved residues in helix VI (P285, E286, Y288, a nd P293) have been substituted. Most of the mutants are enzymatically inactive, Structural perturbations reported by Fourier transform infra red absorption difference spectroscopy of CO adducts of the mutant oxi dases confirm the previous suggestion that this region is adjactent to Cu-B. Furthermore, the analysis of five different substitutions for Y 288 indicates that all lack Cu-B. On the basis of these data, it is pr oposed that Y288 may be a Cu-B ligand along with H333, H334, and H284, and a plausible molecular model of the Cu-B site is presented.