title: Metal reconstitution of particulate methane monooxygenase and heterologous expression of the pmoB subunit.
abstract: Particulate methane monooxygenase (pMMO) is a multisubunit metalloenzyme complex used by methanotrophic bacteria to oxidize methane in the first step of carbon assimilation and energy production. In this chapter, we detail methods to prepare metal free (apo) membrane-bound pMMO and to reconstitute apo pMMO with metal ions. We also describe protocols to clone, express, and refold metal-loaded soluble domain constructs of the pmoB subunit. These approaches were used to address fundamental questions concerning the metal content and location of the pMMO active site.
authors.affiliation: Department of Molecular Biosciences, Northwestern University, Evanston, Illinois, USA.
authors.forename: Stephen M
authors.initials: SM
authors.lastname: Smith
authors.forename: Ramakrishnan
authors.initials: R
authors.lastname: Balasubramanian
authors.forename: Amy C
authors.initials: AC
authors.lastname: Rosenzweig
mesh_terms: D008670:Metals / Q000378:metabolism*; D020619:Methylococcus capsulatus / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D010084:Oxidation-Reduction; D010105:Oxygenases / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D058849:Protein Refolding; D017434:Protein Structure, Tertiary; D021122:Protein Subunits / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012995:Solubility
chemical_list: D008670:Metals; D021122:Protein Subunits; D010105:Oxygenases; C021555:methane monooxygenase