title: Searching for the low affinity ubiquinone binding site in cytochrome bo3from Escherichia coli.
abstract: The cytochrome bo3ubiquinol oxidase is one of three respiratory oxygen reductases in the aerobic respiratory chain of Escherichia coli. The generally accepted model of catalysis assumes that cyt bo3contains two distinct ubiquinol binding sites: (i) a low affinity (QL) site which is the traditional substrate binding site; and (ii) a high affinity (QH) site where a "permanently" bound quinone acts as a cofactor, taking two electrons from the substrate quinol and passing them one-by-one to the heme b component of the enzyme which, in turn, transfers them to the heme o3/CuBactive site. Whereas the residues at the QHsite are well defined, the location of the QLsite remains unknown. The published X-ray structure does not contain quinone, and substantial amounts of the protein are missing as well. A recent bioinformatics study by Bossis et al. [Biochem J. (2014) 461, 305-314] identified a sequence motif G163EFX3GWX2Y173as the likely QLsite in the family of related quinol oxidases. In the current work, this was tested by site-directed mutagenesis. The results show that these residues are not important for catalytic function and do not define the QLsubstrate binding site.
authors.affiliation: Center for Biophysics and Quantitative Biology, University of Illinois, Urbana, IL 61801, USA.
authors.forename: Sylvia K
authors.initials: SK
authors.lastname: Choi
authors.affiliation: Center for Biophysics and Quantitative Biology, University of Illinois, Urbana, IL 61801, USA.
authors.forename: Myat T
authors.initials: MT
authors.lastname: Lin
authors.affiliation: Department of Chemistry, University of Illinois, Urbana, IL 61801, USA.
authors.forename: Hanlin
authors.initials: H
authors.lastname: Ouyang
authors.affiliation: Center for Biophysics and Quantitative Biology, University of Illinois, Urbana, IL 61801, USA; Department of Chemistry, University of Illinois, Urbana, IL 61801, USA; Department of Biochemistry, University of Illinois, Urbana, IL 61801, USA. Electronic address: r-gennis@illinois.edu.
authors.forename: Robert B
authors.initials: RB
authors.lastname: Gennis
mesh_terms: D001665:Binding Sites; D002384:Catalysis; D003573:Cytochrome b Group; D003580:Cytochromes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010084:Oxidation-Reduction; D011485:Protein Binding; D011487:Protein Conformation; D013329:Structure-Activity Relationship; D014451:Ubiquinone / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism*; D014867:Water / Q000378:metabolism
chemical_list: D003573:Cytochrome b Group; D003580:Cytochromes; D029968:Escherichia coli Proteins; C083850:cytochrome bo3, E coli; D014867:Water; D014451:Ubiquinone; C003741:ubiquinol
keywords: Cytochrome bo(3); E. coli; Mutagenesis; Respiration; Ubiquinone