Abstract
Particulate methane monooxygenase (pMMO) is a membrane-bound metalloenzyme that oxidizes methane to methanol in methanotrophic bacteria. Zinc is a known inhibitor of pMMO, but the details of zinc binding and the mechanism of inhibition are not understood. Metal binding and activity assays on membrane-bound pMMO from Methylococcus capsulatus (Bath) reveal that zinc inhibits pMMO at two sites that are distinct from the copper active site. The 2.6 Å resolution crystal structure of Methylocystis species strain Rockwell pMMO reveals two previously undetected bound lipids, and metal soaking experiments identify likely locations for the two zinc inhibition sites. The first is the crystallographic zinc site in the pmoC subunit, and zinc binding here leads to the ordering of 10 previously unobserved residues. A second zinc site is present on the cytoplasmic side of the pmoC subunit. Parallels between these results and zinc inhibition studies of several respiratory complexes suggest that zinc might inhibit proton transfer in pMMO.
Highlights
Particulate methane monooxygenase is a membrane-bound metalloenzyme that oxidizes methane to methanol in methanotrophic bacteria
Metal binding and activity assays on membrane-bound Particulate methane monooxygenase (pMMO) from Methylococcus capsulatus (Bath) reveal that zinc inhibits pMMO at two sites that are distinct from the copper active site
Zinc Inhibition of M. capsulatus (Bath) pMMO—To study the effects of zinc binding on pMMO, membrane extracts were used instead of purified protein because of the low activity of purified preparations [35]
Summary
Particulate methane monooxygenase (pMMO) is a membrane-bound metalloenzyme that oxidizes methane to methanol. Results: Metal binding data and crystal structures reveal that zinc inhibits pMMO at two sites. Metal binding and activity assays on membrane-bound pMMO from Methylococcus capsulatus (Bath) reveal that zinc inhibits pMMO at two sites that are distinct from the copper active site. In the structures of pMMO from M. capsulatus (Bath) and Methylocystis species strain M, this pmoC metal-binding site is occupied by zinc [10]. Previous studies have shown that zinc inhibits pMMO activity in M. capsulatus (Bath) and M. trichosporium OB3b membrane extracts We have used pMMOs from M. capsulatus (Bath) and Methylocystis species strain Rockwell to rigorously address the unresolved questions of how many zinc ions are required to inhibit pMMO, where zinc binds to inhibit pMMO, and whether zinc replaces copper in pMMO
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