Mutagenesis and spectroscopic studies on cytochrome bd quinol oxidase of Escherichia coli

Site-directed mutagenesis studies on the highly conserved residues of cytochrome bd quinol oxidase from Escherichia coli were carried out to investigate their roles in maintaining structure and function of this enzyme. Mutations on two highly conserved residues in subunit I—Glu445 and Arg391 were characterized in detail. Mutant enzymes in which Glu445 was substituted with alanine and glutamine were shown to lose heme b₅₉₅ while retaining the other two hemes—heme b₅₅₈ and heme d. Since no clear role has been discovered for heme b₅₉₅ in the catalytic cycle, the specific knockout of heme b₅₉₅ is a significant step towards understanding its function. Characterizations of mutants on Arg391 suggest that it plays an important role in maintaining the redox potential of heme b₅₅₈ high enough that electrons can be readily transferred from quinol to this heme. Gene fusion studies with the β-lactamase fused to the C-terminal end of the cytochrome bd partial sequence provided conclusive experimental results for a new membrane topology model of subunit I, in which the binuclear center is relocated to the periplasmic side of the membrane. A proton channel now becomes evident so that protons can access the binuclear center and the water resulting from oxygen reduction can be released. Spectroscopic studies of wild type and mutant enzymes using NMR and FTIR were also performed. FTIR spectroscopy showed for the first time the enzyme was able to bind both ubiquinone and menaquinone, and identified an acidic residue, either aspartate or glutamate, as being involved in the binding of ubiquinol/menaquinol. The NMR study was also the first time that high field proton NMR was successfully applied to such a large membrane protein as cytochrome bd with multiple paramagnetic metal centers. The spectra showed that neither heme b₅₉₅ nor heme d undergo spin-state changes upon cyanide binding, and all the hemes retain the same spin-state upon redox state change.