Oxidation of reduced cytochrome c oxidase with 18O2. A search for mu-oxo-bridged metal species in the oxidized enzyme.

We have measured the increase in 18O content of water produced from single turnover oxidations of anerobically reduced cytochrome c oxidase with 18O2 in order to test the hypothesis that a reduced atom of oxygen, originating from dioxygen, remains bound to oxidized cytochrome c oxidase in the form of a mu-oxo-bridge between two metal components when a single turnover occurs. When water samples produced by oxidizing the reduced enzyme with 18O2 were compared to natural abundance control samples obtained by oxidizing with 16O2, all of the 18O2 reduced in a single turnover could be accounted for in the form of additional H218O produced. We conclude that neither atom of the dioxygen reduced is incorporated into the enzyme as a bridge which is stable in the absence of oxidoreductive reactions on the time scale of several minutes.