Cytochrome boa is a respiratory proton-pumping oxygen reductase that is a member of the heme-copper superfamily that utilizes ubiquinol-8 (Q(8)H(2)) as a substrate. The current consensus model has Q(8)H(2) 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 O-2 is reduced to water. The current work shows that the Q(8) 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 bo(3) and that site corresponds to the Q(H) site.