The enzyme mechanism of the Cu-containing small laccase (SLAC) from Streptomyces coelicolor has been investigated by optical and electron paramagnetic resonance spectroscopy. A new intermediate was identified after the reaction of molecular oxygen with the reduced trinuclear site of the type-1-depleted (T1D) form of the enzyme. It has the fingerprint of a biradical with a triplet ground state. One of the spins resides on a Cu in the trinuclear site, tentatively identified as the type-2 site, while the other spin derives from a protein-based radical. The latter is tentatively identified as a tyrosyl radical on the basis of the similarity of the optical characteristics with those observed for a Cu tyrosyl radical pair. The spin-spin distance was found to be 5.0 +/-0.2 angstrom.