The heme-copper oxidase superfamily contains all of the mammalian mitochondrial cytochrome c oxidases, as well as most prokaryotic respiratory oxidases. All members of the superfamily have a subunit homologous to subunit I of the mammalian cytochrome c oxidases. This subunit provides 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 reduced to water. The amino acid sequence of transmembrane helix VI of subunit I is the most highly conserved within the superfamily. Previous efforts have demonstrated that one of the residues in this region, H284, is critical for oxidase activity and for the assembly of CuB. This paper presents the analysis of additional site-directed mutants in which other highly conserved residues in helix VI (P285, E286, Y288, and P293) have been substituted. Most of the mutants are enzymatically inactive. Structural perturbations reported by Fourier transform infrared absorption difference spectroscopy of CO adducts of the mutant oxidases confirm the previous suggestion that this region is adjactent to CuB. Furthermore, the analysis of five different substitutions for Y288 indicates that all lack CuB. On the basis of these data, it is proposed that Y288 may be a CuB ligand along with H333, H334, and H284, and a plausible molecular model of the CuB site is presented.
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