Reaction of the Escherichia coli quinol oxidase cytochrome bo3 with dioxygen: the role of a bound ubiquinone molecule.
We have studied the kinetics of the oxygen reaction of the fully reduced quinol oxidase, cytochrome bo3, using flow-flash and stopped flow techniques. This enzyme belongs to the heme-copper oxidase family but lacks the CuA center of the cytochrome c oxidases. Depending on the isolation procedure, the kinetics are found to be either nearly monophasic and very different from those of cytochrome c oxidase or multiphasic and quite similar to cytochrome c oxidase. The multiphasic kinetics in cytochrome c oxidase can largely be attributed to the presence Of CuA as the donor of a fourth electron, which rereduces the originally oxidized low-spin heme and completes the reduction of O2 to water. Monophasic kinetics would thus be expected, a priori, for cytochrome bo3 since it lacks the CuA center, and in this case we show that the oxygen reaction is incomplete and ends with the ferryl intermediate. Multiphasic kinetics thus suggest the presence of an extra electron donor (analogous to CuA). We observe such kinetics exclusively with cytochrome bo3 that contains a single equivalent of bound ubiquinone-8, whereas we find no bound ubiquinone in an enzyme exhibiting monophasic kinetics. Reconstitution with ubiquinone-8 converts the reaction kinetics from monophasic to multiphasic. We conclude that a single bound ubiquinone molecule in cytochrome bo3 is capable of fast rereduction of heme b and that the reaction with O2 is quite similar in quinol and cytochrome c oxidases.
Puustinen
A
A
Department of Medical Chemistry, Institute of Biomedical Sciences, University of Helsinki, Finland.
Verkhovsky
M I
MI
Morgan
J E
JE
Belevich
N P
NP
Wikstrom
M
M
D001426:Bacterial Proteins / Q000378:metabolism*; D002846:Chromatography, Affinity; D003573:Cytochrome b Group; D003580:Cytochromes / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins; D007700:Kinetics; D010084:Oxidation-Reduction; D010100:Oxygen / Q000378:metabolism*; D010782:Photolysis; D014451:Ubiquinone / Q000378:metabolism*
D001426:Bacterial Proteins; D003573:Cytochrome b Group; D003580:Cytochromes; D029968:Escherichia coli Proteins; C083850:cytochrome bo3, E coli; D014451:Ubiquinone; D010100:Oxygen