Substitutions of charged amino acid residues conserved in subunit I perturb the redox metal centers of the Escherichia coli bo-type ubiquinol oxidase.
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), and serves as a reaction center of the oxidase complex. This work focuses on the functional and structural roles of 14 charged amino acid residues that are conserved in subunit I of the heme-copper terminal oxidases. Substitutions of Lys55, Tyr173, Asp188, Asp256, Arg481, and Arg482 by neutral amino acid residues did not affect the catalytic activity and spectroscopic properties of the cytoplasmic membranes. In contrast, genetic complementation tests indicated that replacements of Arg80, Asp135, Arg257, Glu286, Tyr288, Lys362, Asp407, and Glu540 resulted in nonfunctional enzymes. The R80Q mutation caused loss of a diagnostic peak for low-spin heme b in the 77 K redox difference spectrum. The K362Q, D407N, and E540Q mutations affected the CO-binding by the heme-copper binuclear center. The D135N, R257Q, E286Q, and Y288F mutations specifically eliminated the Cu(B) center from the oxidase complex, whereas the E286D mutant did not show significant perturbations on the redox metal centers even though it was still inactive. Based on these findings and recent crystallographic studies on cytochrome c oxidases, we discuss the possible roles of the conserved charged amino acid residues in subunit I of the heme-copper terminal oxidases.
Kawasaki
M
M
Department of Biological Sciences, Graduate School of Science, The University of Tokyo, Hongo.
Mogi
T
T
Anraku
Y
Y
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000502:physiology*; D002462:Cell Membrane / Q000201:enzymology; D017124:Conserved Sequence; D003300:Copper / Q000032:analysis*; D003573:Cytochrome b Group; D003580:Cytochromes / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins; D005798:Genes, Bacterial / Q000235:genetics; D005816:Genetic Complementation Test; D006418:Heme / Q000032:analysis*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009876:Operon / Q000235:genetics; D010084:Oxidation-Reduction; D017433:Protein Structure, Secondary
D000596:Amino Acids; D003573:Cytochrome b Group; D003580:Cytochromes; D029968:Escherichia coli Proteins; D006418:Heme; D003300:Copper; C017168:cytochrome bo, E coli