Searching for the low affinity ubiquinone binding site in cytochrome bo₃ from Escherichia coli

The cytochrome bo₃ 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 bo₃ contains two distinct ubiquinol binding sites: (i) a low affinity (Q_L) site which is the traditional substrate binding site; and (ii) a high affinity (Q_H) 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/Cu_B active site. Whereas the residues at the Q_H site are well defined, the location of the Q_L 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 G¹⁶³EFX₃GWX₂Y¹⁷³ as the likely Q_L 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_L substrate binding site.