Location of the Substrate Binding Site of the Cytochrome bo₃ Ubiquinol Oxidase from Escherichia coli

Abstract

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.

Role of the tightly bound quinone for the oxygen reaction of cytochrome bo3 oxidase from Escherichia coli

“…Figure shows an overview on the data obtained for mutants at crucial positions in the quinone binding sites and Table summarizes the catalytic potentials and turnover values ( k cat ) determined for the immobilized proteins as described in Materials and methods. For comparison purposes, the turnover numbers reported for the enzymes in solution using the conventional polarographic assay are also shown.…”

“…The DET response depends upon the ability of the enzyme to oxidize the bound UQ 8 , which is retained in these mutants. Interestingly, the ability of the various mutants to stabilize the ubisemiquinone intermediate is not always (see for instance D75H and Q101N in Table ), correlated with the DET response pointing toward a complex role of the radical in catalysis .…”

“…Steady state oxygen reduction specific activities of all the Q101, F93 and D75 mutant enzymes were measured (Table ). The presence of ubiquinone in the protein samples was determined either by extraction followed by HPLC chromatography or by electrochemically induced FTIR difference spectroscopy and corresponds to 1.2–1.7 per protein.…”

“…Subsequently, numerous experiments have suggested that there might be two ubiquinone binding sites in cytochrome bo 3 , a low affinity site and a high affinity site, Q L and Q H . However, recent studies have concluded that there is only one ubiquinone binding site, corresponding to what had been referred to as the high affinity (Q H ) site and it is located in subunit I and can stabilize a ubisemiquinone radical (SQ) .…”

“…). Site‐directed mutations of D75, R71, H98 and I102 resulted in the total loss of the quinol oxidase activity , whereas most of Q101 mutants retain a partial activity . D75 has been suggested to act as a proton acceptor from the quinol .…”