Biophysical and structural characterization of particulate methane monooxygenase from Methylococcus capsulatus (Bath)

Particulate methane monooxygenase (pMMO) is a three-subunit integral membrane metalloenzyme that converts methane to methanol under ambient conditions. Although pMMO is the predominant methane oxidation catalyst in nature, it has proved difficult to isolate, and the literature addressing details of its structure and active site composition has been mired in controversy for longer than a decade. Knowledge of how pMMO activates the inert methane C--H bond is of fundamental chemical interest, and could lead to development of new synthetic catalysts that could impact the use of methane (natural gas) as an alternative energy source.;This dissertation describes biochemical, biophysical and crystallographic studies that contribute significantly to our molecular understanding of pMMO. Protocols were developed for the purification of pMMO from the membranes of Methylococcus capsulatus (Bath). Enzyme activity was detected for purified pMMO. As determined by gel electrophoresis, chemical crosslinking, and analytical ultracentrifugation, the overall molecular mass of pMMO is consistent with a 200 kDa species, possibly corresponding to a pMMO dimer, with additional higher order oligomers observed.;Purified pMMO contains 2--3 copper and ∼1 iron ions per monomer, which were probed by electron paramagnetic resonance (EPR), ultraviolet-visible-near-infrared, and X-ray absorption (XAS) spectroscopy. The copper ions are present in a mixture of Cu(I) and Cu(II). EPR spectroscopic parameters indicate type 2 mononuclear copper, and XAS studies have provided the first direct evidence for a copper-containing cluster with a 2.57 A Cu-Cu interaction in pMMO.;Finally, after extensive screening and optimization, large, well-ordered crystals of pMMO for structure determination were obtained. The refined 2.8 A resolution crystal structure provides a wealth of new insight into pMMO, including details of three distinct metal centers and their locations within the overall protein fold. Two metal centers, modeled as mononuclear and dinuclear copper, reside in the soluble region of the protein. A third metal center, occupied by zinc in the crystal, is located within the membrane. This work lays the foundation for future directed biochemical and mechanistic studies of pMMO.