Kinetic measurements on single-turnover processes in laccase established fast type-1 Cu to trinudear Cu cluster (TNC) intramolecular electron transfer (IET) in the reduction of the native intermediate (NI), the fully oxidized form of the enzyme formed immediately after O-O bond cleavage in the mechanism of O-2 reduction. Alternatively, slow IET kinetics was observed in the reduction of the resting enzyme, which involves proton-coupled electron transfer on the basis of isotope measurements. The >10(3) difference between the IET rates for these two processes confirms that the NI, rather than the resting enzyme that has been defined by crystallography, is the fully oxidized form of the TNC in catalytic turnover. Computational modeling showed that reduction of NI is fast because of the larger driving force associated with a more favorable proton affinity of its mu(3)-oxo moiety generated by reductive cleavage of the O-O bond. This defines a unifying mechanism in which reductive cleavage of the O-O bond is coupled to rapid JET in the multicopper oxidases.