The interactions of cytochrome bo3 from Escherichia coli with its substrates - ubiquinone and oxygen

Heme-copper respiratory oxygen reductases reduce O2 to water and use the redox free energy to generate the proton motive force. Among the heme-copper oxygen reductases are the quinol oxidases, which catalyze the 2-electron oxidation of ubiquinol or menaquinol instead of cytochrome c. Escherichia coli (E. coli) cytochrome bo3 is the best characterized quinol oxidase. Depending on the detergent used to solubilize the enzyme, cyt bo3, preparations of this enzyme contain between 0 to 2 equivalents of ubiquinone-8. Studies using ubiquinol-1, a soluble quinol substrate, indicate one high affinity quinone binding site, which acts as a non-exchanging co-factor, and a second, low-affinity site which is the substrate site. The residues interacting with the quinone at the high affinity QH site are well defined through numerous mutagenesis and spectroscopic studies, but the identification of the low affinity QL site has remained difficult. To clarify whether the low affinity QL exists, we used structural models to guide us. First is based on the new structure of cytochrome bo3 we have obtained by collaboration with the Stowell group. It shows two ubiquinone molecules bound to the enzyme. The benzoquinone headgroup of one bound quinone is located at the expected QH site. The headgroup of the second bound quinone is, however, located on the opposite side of the molecule. Site-directed mutagenesis of residues located at the putative second site do not indicate that this second site is functionally important, leaving the question of the existence of the QL site open. The second model is based on bioinformatics. Recent publication from Bossis group used bioinformatics methods to identify a possible QL site. We have tested this model using site-directed mutagenesis but have found no evidence for disruption of a substrate binding site. Along with these two models, we have revisited the possibility of a QL site in subunit II with site-directed mutagenesis but did not find the QL site. A strong argument in favor of the existence of ...