The crystal structure of the particulate methane monooxygenase (pMMO) from Methylococcus capsulatus (Bath) has been reported recently [Lieberman, R. L., and Rosenzweig, A. C. (2005) Crystal structure of a membrane-bound metalloenzyme that catalyses the biological oxidation of methane, Nature 434, 177-182]. Subsequent work has shown that the preparation on which the X-ray analysis is based might be missing many of the important metal cofactors, including the putative trinuclear copper cluster at the active site as well as ca. 10 copper ions (E-clusters) that have been proposed to serve as a buffer of reducing equivalents to re-reduce the copper atoms at the active site following the catalytic chemistry [Chan, S. I., Wang, V. C.-C., Lai, J. C.-H., Yu, S. S.-F., Chen, P. P.-Y., Chen, K. H.-C., Chen, C.-L., and Chan, M. K. (2007) Redox potentiometry studies of particulate methane monooxygenase: Support for a trinuclear copper cluster active site, Angew. Chem., Int. Ed. 46, 1992-1994]. Since the aqueous-exposed domains of the 45 kDa subunit (PmoB) have been suggested to be the putative binding domains for the E-cluster copper ions, we have cloned and overexpressed in Escherichia coli the two aqueous-exposed subdomains toward the N- and C-termini of the subunit: the N-terminal subdomain (residues 54-178) and the C-terminal subdomain (residues 257-394 and 282-414). The recombinant C-terminal water-exposed subdomain is shown to behave like a Cu(I) sponge, taking up to ca. 10 Cu(I) ions cooperatively when cupric ions are added to the protein fragment in the presence of dithiothreitol or ascorbate. In addition, circular dichroism measurements reveal that the C-terminal subdomain folds into a beta-sheet structure in the presence of Cu(I). The propensity for the C-terminal subdomain to bind Cu(I) is consistent with the high redox potential(s) determined for the E-cluster copper ions in the pMMO. These properties of the E-clusters are in accordance with the function proposed for these copper ions in the turnover cycle of the enzyme.