Biophysical and structural characterization of particulate methane monooxygenase and methanobactin from Methylosinus trichosporium OB3b

Particulate methane monooxygenase (pMMO) is a membrane-bound metalloenzyme that oxidizes methane to methanol in methanotrophic bacteria. The nature of the pMMO active site and the overall metal content are controversial, with spectroscopic and crystallographic data suggesting the presence of a mononuclear copper center, a dinuclear copper center, a trinuclear center, and a diiron center or combinations thereof in various samples. Most studies have focused on pMMO from Methylococcus capsulatus (Bath). In this work, pMMO from a second organism, Methylosinus trichosporium OB3b, has been purified and characterized by spectroscopic and crystallographic methods. Purified M. trichosporium OB3b pMMO contains ∼2 copper ions per 100 kDa protomer. Electron paramagnetic resonance (EPR) spectroscopic parameters indicate type 2 Cu(II) is present as two distinct species. Extended X-ray absorption fine structure (EXAFS) data are best fit with oxygen/nitrogen ligands and a Cu-Cu interaction at 2.52 A.; The crystal structure of M. trichosporium OB3b was solved to 3.9 A resolution. Crystallographic characterization of M. trichosporium OB3b pMMO shows that a mononuclear copper center found in the M. capsulatus (Bath) pMMO X-ray structure is not present whereas a dinuclear copper center is clearly conserved. Notably, a metal center occupied by zinc in the M. capsulatus (Bath) pMMO structure is occupied by copper in M. trichosporium OB3b pMMO. These findings extend previous work on pMMO from M. capsulatus (Bath) and provide new insight into the functional importance of the different metal centers.; The oxidation state of copper bound to methanobactin, a small siderophore-like molecule from M. trichosporium OB3b, was also investigated. Purified methanobactin loaded with Cu(II) exhibits a weak EPR signal probably due to adventitious Cu(II). The EPR signal intensity increases significantly upon addition of the strong oxidant nitric acid. Features of the X-ray absorption near edge spectrum, including a 1s→4p transition at 8985.5 eV, further indicate the presence of Cu(I). EXAFS data were best fit using a multiple scattering model generated from previously reported crystallographic parameters. These results establish definitively that M. trichosporium OB3b methanobactin binds Cu(I) and suggest that methanobactin itself reduces Cu(II) to Cu(I).