Methane hydroxylation at the mononuclear and dinuclear copper sites of pMMO is discussed using quantum mechanical and QM/MM calculations. Possible mechanisms are proposed with respect to the formation of reactive copper-oxo and how they activate methane. Dioxygen is incorporated into the Cu-I species to give a Cu-II- superoxo species, followed by an H-atom transfer from a tyrosine residue near the monocopper active site. A resultant CuII- hydroperoxo species is next transformed into a Cu-III-oxo species and a water molecule by the abstraction of an H-atom from another tyrosine residue. This process is accessible in energy under physiological conditions. Dioxygen is also incorporated into the dicopper site to form a (mu-eta(2):eta(2)-peroxo)dicopper species, which is then transformed into a bis(mu-oxo) dicopper species. The formation of this species is more favorable in energy than that of the monocopper- oxo species. The reactivity of the Cu-III-oxo species is sufficient for the conversion of methane to methanol if it is formed in the protein environment. Since the sigma* orbital localized in the Cu-O bond region is singly occupied in the triplet state, this orbital plays a role in the homolytic cleavage of a C-H bond of methane. The reactivity of the bis(mu-oxo) dicopper species is also sufficient for the conversion of methane to methanol. The mixed-valent bis(mu-oxo)(CuCuIII)-Cu-II species is reactive to methane because the amplitude of the sigma* singly occupied MO localized on the bridging oxo moieties plays an essential role in C-H activation.