Substitution of asparagine for aspartate-135 in subunit I of the cytochrome bo ubiquinol oxidase of Escherichia coli eliminates proton-pumping activity.
The terminal quinol oxidase, cytochrome bo, of Escherichia coli is a member of the large terminal oxidase family, which includes cytochrome aa3-type terminal oxidases from bacteria, plants, and animals. These enzymes conserve energy by linking electron transfer to vectorial proton translocation across mitochondrial or bacterial cell membranes. Site-directed mutagenesis of the five most highly conserved acidic amino acids in subunit I of cytochrome bo was performed to study their role in proton transfer. Mutation of only one of these sites, Asp135, to the corresponding amide, results in a dramatic decrease in proton pumping but with little change in electron-transfer activity. However, the conservative mutation Asp135Glu is active in proton translocation. It is proposed that an acidic residue at position 135 in subunit I may be important to form a functional proton input channel of the proton pump.
School of Chemical Sciences, University of Illinois, Urbana 61801.
J W
JW
Thomas
A
A
Puustinen
J O
JO
Alben
R B
RB
Gennis
M
M
Wikström
D000595:Amino Acid Sequence; D001216:Asparagine; D001224:Aspartic Acid; D003001:Cloning, Molecular; D017124:Conserved Sequence; D003576:Electron Transport Complex IV / Q000737:chemistry* / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D046911:Macromolecular Substances; D008961:Models, Structural; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D017550:Spectroscopy, Fourier Transform Infrared / Q000379:methods
D046911:Macromolecular Substances; D011994:Recombinant Proteins; D001224:Aspartic Acid; D001216:Asparagine; C038862:cytochrome o oxidase; D003576:Electron Transport Complex IV