X-RAY-ABSORPTION AND EPR STUDIES ON THE COPPER IONS ASSOCIATED WITH THE PARTICULATE METHANE MONOOXYGENASE FROM METHYLOCOCCUS-CAPSULATUS (BATH) - CU(I) IONS AND THEIR IMPLICATIONS

Parallel X-ray absorption edge and EPR studies of the particulate meth ane monooxygenase in situ reveal that the enzyme contains unusually hi gh levels of copper ions with a significant portion of the copper ions existing as Cu(I) in the ''as-isolated'' form (70-80%). The observati on of high levels of reduced copper in a monooxygenase is surprising c onsidering that the natural cosubstrate of the enzyme is dioxygen. Tow ard clarifying the roles of the various copper ions in the enzyme, we have successfully prepared different states of the protein in the memb rane-bound form at various levels of reduction using dithionite, dioxy gen, and ferricyanide. EPR intensity analysis of the fully-oxidized pr eparations indicates that the bulk of copper ions are arranged in clus ter units. The fully-reduced protein obtained by reduction by dithioni te has been used to initiate the single turnover of the enzyme in the presence of dioxygen. Differential reactivity toward dioxygen was obse rved upon analyzing the copper reduction levels in these synchronized preparations. The enzyme is capable of supporting turnover in the abse nce of external electron donors in the highly reduced states. These re sults suggest the presence of at least two classes of copper ions in t he particulate methane monooxygenase. As a working hypothesis, we have referred to these classes of copper ions as (1) the catalytic (C) clu sters, which function principally as the catalytic core of the enzyme, and (2) the electron-transfer (E) clusters, which are presumed to be the source of endogenous reducing equivalents and therefore function i n an electron-transfer capacity.