Substitutions of conserved aromatic amino acid residues in subunit I perturb the 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 CuB, 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 at the boundary of the membrane) of subunit I. Sixteen aromatic amino acid residues were individually substituted by Leu, except for Tyr61 and Trp282 by Phe and Phe415 by Trp. Leu substitutions of Trp280 and Tyr288 in helix VI, Trp331 in loop VII-VIII, and Phe348 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.
Mogi
T
T
Department of Biological Sciences, Graduate School of Science, University of Tokyo, Japan. mogi@biol.s.u-tokyo.ac.jp
Minagawa
J
J
Hirano
T
T
Sato-Watanabe
M
M
Tsubaki
M
M
Uno
T
T
Hori
H
H
Nakamura
H
H
Nishimura
Y
Y
Anraku
Y
Y
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics*; D000598:Amino Acids, Cyclic / Q000235:genetics*; D002384:Catalysis; D017124:Conserved Sequence; D003300:Copper / Q000378:metabolism*; D003486:Cyanides / Q000378:metabolism; D004578:Electron Spin Resonance Spectroscopy; D003576:Electron Transport Complex IV / Q000235:genetics* / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D006418:Heme / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D011485:Protein Binding / Q000235:genetics; D013056:Spectrophotometry, Ultraviolet; D017550:Spectroscopy, Fourier Transform Infrared; D013059:Spectrum Analysis, Raman
D000598:Amino Acids, Cyclic; D003486:Cyanides; D006418:Heme; D003300:Copper; C038862:cytochrome o oxidase; D003576:Electron Transport Complex IV