The PmoB subunit of particulate methane monooxygenase (pMMO) in Methylococcus capsulatus (Bath): The CuI sponge and its function

In this study, we describe efforts to clarify the role of the copper cofactors associated with subunit B (PmoB) of the particulate methane monooxygenase (pMMO) from Methylococcus capsulatus (Bath) (M. capsulatus). This subunit exhibits strong affinity toward Cu-I ions. To elucidate the high copper affinity of the subunit, the full-length PmoB, and the N-terminal truncated mutants PmoB(33-414) and PmoB(55-414), each fused to the maltosebinding protein (MBP), are cloned and over-expressed into Escherichia coli (E. coli) K12 TB1 cells. The Y374F, Y374S and M300L mutants of these protein constructs are also studied. When this E. colt is grown with the pmoB gene in 1.0 mM Cu-II, it behaves like M. capsulatus (Bath) cultured under high copper stress with abundant membrane accumulation and high Cu-I content. The recombinant PmoB proteins are verified by Western blotting of antibodies directed against the MBP sub-domain in each of the copper-enriched PmoB proteins. Cu K-edge Xray absorption near edge spectroscopy (XANES) of the copper ions confirms that all the PmoB recombinants are Cu-I proteins. All the PmoB proteins show evidence of a dicopper site according to analysis of the Cu extended X-ray absorption edge fine structure (EXAFS) of the membranes. No specific activities toward methane and propene oxidation are observed with the recombinant membrane-bound PmoB proteins. However, significant production of hydrogen peroxide is observed in the case of the PmoB(33-414) mutant. Reaction of the dicopper site with dioxygen produces hydrogen peroxide and leads to oxidation of the Cu-I ions residing in the C-terminal subdomain of the PmoB subunit.