REACTION OF THE ESCHERICHIA-COLI QUINOL OXIDASE CYTOCHROME BO(3) WITHDIOXYGEN - THE ROLE OF A BOUND UBIQUINONE MOLECULE

We have studied the kinetics of the oxygen reaction of the fully reduc ed quinol oxidase, cytochrome bo(3), 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 i solation procedure, the kinetics are found to be either nearly monopha sic and very different from those of cytochrome c oxidase or multiphas ic 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 O-2 to water. Mo nophasic kinetics would thus be expected, a priori, for cytochrome bo( 3) since it lacks the CUA center, and in this case we show that the ox ygen reaction is incomplete and ends with the ferryl intermediate. Mul tiphasic kinetics thus suggest the presence of an extra electron donor (analogous to CUA) We observe such kinetics exclusively with cytochro me bo(3) that contains a single equivalent of bound ubiquinone-8, wher eas we find no bound ubiquinone in an enzyme exhibiting monophasic kin etics. Reconstitution with ubiquinone-l converts the reaction kinetics from monophasic to multiphasic. We conclude that a single bound ubiqu inone molecule in cytochrome bo(3) is capable of fast rereduction of h eme b and that the reaction with O-2 is quite similar in quinol and cy tochrome c oxidases.