THE NATURE OF THE COPPER IONS IN THE MEMBRANES CONTAINING THE PARTICULATE METHANE MONOOXYGENASE FROM METHYLOCOCCUS-CAPSULATUS (BATH)

It is shown that the particulate methane monooxygenase (pMMO) has an o bligate requirement for copper. The MMO activity in the particulate fr actions obtained from Methylococcus capsulatus (Bath) cells is found t o increase with increasing copper content of the membranes. The enzyme activity from membranes obtained from cells grown at low copper level s can be stimulated further by the addition of Cu(II) ions to the assa y medium. The membrane-bound copper ions can exist in both Cu(II) and Cu(I) forms. EPR and magnetic susceptibility characterizations of the membranes indicate the presence of an exchange-coupled trinuclear Cu(I I) cluster when the bulk of the copper ions is oxidized. However, the functional form of the enzyme is the reduced or partially reduced form . The copper ions in the membrane fractions as isolated often exhibit a high level of reduction. An EPR spectrum with one unpaired electron spin delocalized over three copper nuclei has been observed for the tw o-electron reduced trinuclear copper cluster. The high correlation bet ween the copper level in the membranes and enzymatic activity as well as the high reactivity of the reduced copper clusters toward dioxygen strongly indicate that the membrane-bound copper ions constitute the a ctive sites of the pMMO.