Location of the Substrate Binding Site of the Cytochrome <i>bo</i>₃ Ubiquinol Oxidase from <i>Escherichia coli</i>

Abstract

Cytochrome bo₃ is a respiratory proton-pumping oxygen reductase that is a member of the heme-copper superfamily that utilizes ubiquinol-8 (Q₈H₂) as a substrate. The current consensus model has Q₈H₂ oxidized at a low affinity site (Q_L), passing electrons to a tightly bound quinone cofactor at a high affinity site (Q_H site) that stabilizes the one-electron reduced ubisemiquinone, facilitating the transfer of electrons to the redox active metal centers where O₂ is reduced to water. The current work shows that the Q₈ bound to the Q_H site is more dynamic than previously thought. In addition, mutations of residues at the Q_H site that do not abolish activity have been re-examined and shown to have properties expected of mutations at the substrate binding site (Q_L): an increase in the K_M of the substrate ubiquinol-1 (up to 4-fold) and an increase in the apparent K_i of the inhibitor HQNO (up to 8-fold). The data suggest that there is only one binding site for ubiquinol in cyt bo₃ and that site corresponds to the Q_H site.