Location of the Substrate Binding Site of the Cytochrome bo Ubiquinol Oxidase from Escherichia coli.

Cytochrome bo is a respiratory proton-pumping oxygen reductase that is a member of the heme-copper superfamily that utilizes ubiquinol-8 (QH) as a substrate. The current consensus model has QH oxidized at a low affinity site (Q), passing electrons to a tightly bound quinone cofactor at a high affinity site (Q site) that stabilizes the one-electron reduced ubisemiquinone, facilitating the transfer of electrons to the redox active metal centers where O is reduced to water. The current work shows that the Q bound to the Q site is more dynamic than previously thought. In addition, mutations of residues at the Q site that do not abolish activity have been re-examined and shown to have properties expected of mutations at the substrate binding site (Q): an increase in the K of the substrate ubiquinol-1 (up to 4-fold) and an increase in the apparent K of the inhibitor HQNO (up to 8-fold). The data suggest that there is only one binding site for ubiquinol in cyt bo and that site corresponds to the Q site.