Formation of an active oxygen species at the dicopper site of pMMO is studied by using density functional theory (DFT) calculations. The role of the amino acid residues of tyrosine (Tyr374) and glutamate (Glu35) located in the second coordination sphere of the dicopper site is discussed in detail. The phenolic proton of the tyrosine residue is transferred to the Cu2O2 core in a two-step manner via the glutamate residue, and an electron is directly transferred to the Cu2O2 core. These proton- and electron-transfer processes induce the O-O bond cleavage of the μ-η2:η2-peroxodicopper(II) species to form the (μ-oxo)(μ-hydroxo)CuIICuIII species, which is able to play a key role of methane hydroxylation at the dicopper site of pMMO ( Inorg. Chem. 2013 , 52 , 7907 ). This proton-coupled electron-transfer mechanism is a little different from that in tyrosinase in that the proton of substrate tyrosine is directly transferred to the dicopper site ( J. Am. Chem. Soc. 2006 , 128 , 9873 ) because there is no proton acceptor in the vicinity of the dicopper site of tyrosinase. The rate-determining step for the formation of the (μ-oxo)(μ-hydroxo)CuIICuIII species is determined to be the O-O bond cleavage. These results shed new light on the interpretation of the role of the tyrosine and glutamate residues located in the second coordination sphere of the dicopper site of pMMO.