title: Structural features of covalently cross-linked hydroxylase and reductase proteins of soluble methane monooxygenase as revealed by mass spectrometric analysis.
abstract: Soluble methane monooxygenase requires complexes between its three component proteins for efficient catalysis. The hydroxylase (MMOH) must bind both to the reductase (MMOR) and to the regulatory protein (MMOB) to facilitate oxidation of methane to methanol. Although structures of MMOH, MMOB, and one domain of MMOR have been determined, less geometric information is available for the complexes. To address this deficiency, MMOH and MMOR were cross-linked by a carbodiimide reagent and analyzed by specific proteolysis, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, and capillary high performance liquid chromatography mass spectrometry. Tandem mass spectra conclusively identified two amine-to-carboxylate cross-linked sites involving the alpha subunit of MMOH and the [2Fe-2S] domain of MMOR (MMOR-Fd). In particular, the N terminus of the MMOH alpha subunit forms cross-links to the side chains of MMOR-Fd residues Glu-56 and Glu-91. These Glu residues are close to one another on the surface of MMOR-Fd and >25 A from the [2Fe-2S] cluster. Because the N terminus of the alpha subunit of MMOH was not located in the crystal structure of MMOH, a detailed structural model of the complex based on the cross-link was precluded; however, a previously proposed binding site for MMOR on MMOH could be ruled out. Based on the cross-linking results, a MMOR E56Q/E91Q double mutant was generated. The mutant retains >80% of MMOR NADH oxidase activity but reduces sMMO activity to approximately 65% of the level supported by the wild type reductase. Cross-linking to MMOH was diminished but not abolished in the double mutant, indicating that other residues of MMOR also form cross-links to MMOH.
authors.affiliation: Department of Chemistry, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA.
authors.forename: Daniel A
authors.initials: DA
authors.lastname: Kopp
authors.forename: Eric A
authors.initials: EA
authors.lastname: Berg
authors.forename: Catherine E
authors.initials: CE
authors.lastname: Costello
authors.forename: Stephen J
authors.initials: SJ
authors.lastname: Lippard
mesh_terms: D001665:Binding Sites; D003432:Cross-Linking Reagents / Q000737:chemistry; D003461:Crystallography; D020619:Methylococcus capsulatus; D020602:Methylosinus trichosporium; D006899:Mixed Function Oxygenases / Q000737:chemistry*; D010088:Oxidoreductases / Q000737:chemistry*; D010105:Oxygenases / Q000737:chemistry*; D017434:Protein Structure, Tertiary; D012995:Solubility; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D003432:Cross-Linking Reagents; D006899:Mixed Function Oxygenases; D010088:Oxidoreductases; D010105:Oxygenases; C021555:methane monooxygenase