Searching for the low affinity ubiquinone binding site in cytochrome bo 3 from Escherichia coli

• Published in: Biochimica et biophysica acta. Bioenergetics Vol. 1858; no. 5; p. 366
• Main Authors: Choi, Sylvia K, Lin, Myat T, Ouyang, Hanlin, Gennis, Robert B
• Format: Journal Article
• Language: English
• Published: Netherlands 01-05-2017
• Subjects: Binding Sites; Catalysis; Cytochromes - chemistry; Cytochromes - genetics; Cytochromes - metabolism; Escherichia coli - enzymology; Escherichia coli - genetics; Escherichia coli Proteins - chemistry; Escherichia coli Proteins - genetics; Escherichia coli Proteins - metabolism; Models, Molecular; Mutagenesis, Site-Directed; Mutation; Oxidation-Reduction; Protein Binding; Protein Conformation; Structure-Activity Relationship; Ubiquinone - analogs & derivatives; Ubiquinone - chemistry; Ubiquinone - metabolism; Water - metabolism; Ubiquinone; Mutagenesis; Cytochrome bo; Respiration; E. coli
• ISSN: 0005-2728

The cytochrome bo
ubiquinol 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 bo
contains two distinct ubiquinol binding sites: (i) a low affinity (Q
) site which is the traditional substrate binding site; and (ii) a high affinity (Q
) 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 o
/Cu
active site. Whereas the residues at the Q
site are well defined, the location of the Q
site 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 G
EFX
GWX
Y
as the likely Q
site 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 Q
substrate binding site.